Plants containing a cytosolic acetyl CoA-carboxylase nucleic acid

ABSTRACT

Plants are disclosed that contain a recombinant nucleic acid construct comprising a nucleic acid encoding a cytosolic acetyl coA-carboxylase (ACCase) operably linked to a promoter. Seeds produced from such plants exhibit statistically significantly increased oil content as compared to seeds produced by a corresponding plant lacking the nucleic acid encoding the ACCase. Methods of producing seeds exhibiting statistically significantly increased oil content are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119(e) of U.S.provisional application serial No. 60/198,794, filed Apr. 20, 2000.

TECHNICAL FIELD

[0002] This invention relates to oilseed plants, and more particularlyto plants containing a nucleic acid encoding a cytosolic acetylcoA-carboxylase (ACCase) enzyme.

BACKGROUND

[0003] Acetyl-CoA carboxylase [ACCase; EC 6.4.1.2] catalyzes the firstcommitted step in fatty acid biosynthesis by converting acetyl-CoA tomalonyl-CoA. In plants, a multisubunit (MS) form and a multifunctional(MF) form of ACCase have been identified. The MS form is composed ofdissociable subunits of different sizes, including a biotin carboxylcarrier protein (BCCP), α- and β-carboxyltransferases (α-CT and β-CT,respectively), and a biotin carboxylase (BC). The MS form is present inplastids of dicotyledenous and of non-Gramineae monocotyledenous plantsand is primarily involved in the biosynthesis of fatty acids.

[0004] The MF form of a plant ACCase is similar to mammalian ACCase (andis sometimes designated “eukaryotic” or “cytosolic” ACCase), in that itis a MF polypeptide with a molecular weight of more than 200 kDa. The MFform of ACCase from plants contains BCCP, BC, α-CT and β-CT functionaldomains in a single polypeptide. MF ACCase is most likely present in thecytosol of all plant species and in the chloroplasts of Gramineaeplants. Plant MF ACCase is involved in the biosynthesis of very longchain fatty acids, flavonoids, and in the malonation of amino acids andaminocyclopropane-1-carboxylate (a precursor to ethylene).

[0005] Antisense nucleic acids against an MF ACCase have been introducedinto Brassica napus (White et al., 1998, in Adv. in Plant Lipid Res.,pp. 62-66, eds., Sánchez, J., Cerdá-Olmedo, E. & Martinez-Horce, E.,Universidad De Sevilla, Spain) and an Arabidopsis genomic DNA encodingan MF ACCase under the control of a napin seed-specific promoter andlinked to a small subunit (ss) Rubisco transit peptide was introducedinto B. napus (Roesler et al., 1997, Plant physiol., 113:75-81; U.S.Pat. No. 5,925,805).

SUMMARY

[0006] Plants have been engineered to express a nucleic acid encoding anMF acetyl coA-carboxylase (ACCase), hereinafter referred to as cytosolicACCase. Oil content was significantly increased in plants containing thecytosolic ACCase coding sequences.

[0007] In general, the invention feature plants containing a nucleicacid construct carrying a nucleic acid encoding a cytosolic ACCaseoperably linked to a promoter and lacking a transit peptide. This plantproduces seeds that exhibit a statistically significant increase in oilcontent as compared to seeds produced by a corresponding plant lackingsuch a construct.

[0008] The invention additionally features plants containing a nucleicacid construct carrying a nucleic acid encoding a cytosolic ACCaselacking introns operably linked to a promoter. This plant produces seedsthat exhibit a statistically significant increase in oil content ascompared to seeds produced by a corresponding plant lacking such aconstruct.

[0009] The invention also features methods of producing a transgenicplant. This method includes selecting progeny transgenic plants of aplant containing a nucleic acid construct carrying a nucleic acidencoding a cytosolic ACCase operably linked to a promoter. Following atleast one generation of selection, one or more of the progeny transgenicplants produce seeds exhibiting a statistically significant increase inoil content as compared to seeds produced by a corresponding plantlacking such a construct.

[0010] The invention further features methods of producing a plant byintroducing a construct carrying a nucleic acid encoding a cytosolicACCase operably linked to a promoter into one or more plants. Progeny ofthese plants, following at least one generation of selection, produceseeds that exhibit a statistically significant increase in oil contentwhen compared to seeds produced by a corresponding plant lacking such aconstruct.

[0011] Yet another feature of the invention are methods of increasingthe oil content in seeds by creating a plant containing a nucleic acidconstruct carrying a gene encoding a cytosolic ACCase operably linked toa promoter; and selecting progeny of the plant that exhibit astatistically significant increase in oil content in seeds as comparedto seeds produced by a corresponding plant lacking such a construct.

[0012] Additionally featured in the invention are seeds produced by theabove-described plants, and progeny of those plants, wherein the progenyproduce seeds that exhibit a statistically significant increase in oilcontent when compared to seeds produced by the progeny of plants lackingsuch a construct.

[0013] Typically, the increase in oil content is from about 5% to about25% on a dry weight basis. The above-described selection steps caninclude selecting progeny that contain the nucleic acid construct.Generally, soybean plants or Brassica plants, for example, Brassicanapus, B. rapa, B. juncea, B. carinata, B. nigra and B. oleracea areuseful in the invention.

[0014] Still yet another feature of the invention is a nucleic acidconstruct carrying a cytosolic ACCase coding sequence operably linked toa promoter but lacking a transit peptide and a nucleic acid constructcarrying a cytosolic ACCase coding sequence lacking introns operablylinked to a promoter.

[0015] A promoter included in a construct of the invention can be acauliflower mosaic virus (CaMV) 35S promoter. Unless otherwiseindicated, the ACCase constructs described herein may or may not includenucleic acid sequences encoding a transit peptide operably linked to thenucleic acid sequences encoding the cytosolic ACCase. An example of atransit peptide is a tobacco small subunit Rubisco transit peptide. Inaddition, a nucleic acid encoding a cytosolic ACCase can encode a plantcytosolic ACCase, for example, an alfalfa cytosolic ACCase. Further andunless otherwise indicated, a nucleic acid encoding the ACCase can lackintrons.

[0016] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. The materialsmethods, and examples are illustrative only and not intended to belimiting. Suitable methods and materials are described below, althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol.

[0017] The details of one or more embodiments of the invention are setforth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages of the invention will be apparent fromthe drawings and detailed description, and from the claims.

DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 is the nucleotide and amino acid sequence (SEQ ID NO:3 and4 respectively) of the tobacco small subunit (ss) Rubisco transitpeptide and 5′ portion of the mature ss Rubisco protein (underlined).

[0019]FIG. 2 is a representative +6ACCase construct (SEQ ID NO:5). Thenucleotide sequence encoding a transit peptide and the 5′ portion of asmall subunit (ss) Rubisco gene from tobacco is shown operably linked toan alfalfa cytosolic ACCase coding sequence. A consensus sequence forinitiation of translation is italicized and includes the 3′ end of a 35Scauliflower mosaic virus (CaMV) promoter and the 5′ sequence encodingthe tobacco ssRubisco transit peptide. The ACCase sequence correspondsto a portion of the coding sequence and 3′ untranslated sequences (SeeGenbank Accession No. L25042); for the entire ACCase coding sequence).Arrows indicate the methionine-initiated (M) start codon of thessRubisco transit peptide, the beginning of the portion of the ssRubiscomature protein included in the construct, the beginning and end of theACCase coding sequence as published in GenBank, and the end of theACCase 3′ untranslated sequences. The BamHI and KpnI restriction siteswere used to clone the +6ACCase construct into the ptet vector.

[0020]FIG. 3 is a representative −7ACCase construct (SEQ ID NO:6). Theitalicized consensus sequence for the initiation of translation includesthe 3′ end of a 35S cauliflower mosaic virus (CaMV) promoter and the 5′portion of an alfalfa cytosolic acetyl coA-carboxylase (ACCase) codingsequence (Shorrosh et al., 1994). The ACCase sequences are as describedin the legend to FIG. 2. Arrows indicate the methionine-initiated (M)start codon, the end of the ACCase coding as published in GenBank andthe end of the ACCase 3′ untranslated sequences. The BamHI and KpnIrestriction sites were used to clone the −7ACCase construct into theptet vector.

[0021]FIG. 4 is the nucleotide and amino acid sequence (SEQ ID NO:7 and8, respectively) of an alfalfa cytosolic acetyl coA-carboxylase (ACCase)(GenBank Accession No. L25042 plus additional 3′ untranslatedsequences).

[0022] Like reference symbols in the various drawings indicate likeelements.

DETAILED DESCRIPTION

[0023] All percent oil content and percent protein content are reportedbased upon dry weight. As used herein, “oil content” or “percent oilcontent” refers to the oil content in a particular tissue. “Oils” aretypically triacylglycerols. Oil content can be measured in by NMR (usingAmerican Oil Chemists' Society (AOCS) Method AM 2-93 and AOCSRecommended Practice AK 4-95) or by NIR (using AOCS Method AK 3-94 andAOCS Procedure AM 1-92).

[0024] As used herein, “protein content” or “percent protein content”refers to the protein content in a particular tissue. The proteincontent in seeds typically includes storage proteins, as well as otherpeptide/polypeptide components. Protein content can be determined by NIR(using AOCS Method BA 4e-93).

[0025] As used herein, “high oleic acid” refers to an oleic acid(C_(18:1)) content in seeds greater than 70% based on total fatty acidcomposition after hydrolysis. A typical high oleic Brassica lineexhibits an oleic acid content of at least 70%; for example, an oleicacid content of about 80%, or about 90% based on total fatty acidcomposition after hydrolysis. Oleic acid is typically measured by gaschromatography (GC) using AOCS Method Ce 1e-91.

[0026] As used herein, “high erucic acid” refers to an erucic acid(C_(22:1)) content greater than 45% based on total fatty acidcomposition after hydrolysis. A typical high erucic acid Brassica linewould exhibit an erucic acid content of at least 45%; for example, anerucic acid content of 50%, 55% or even greater based on total fattyacid composition after hydrolysis. Erucic acid is typically measured byGC using AOCS Method Ce 1e-91.

[0027] As used herein, “FDA saturated fatty acid content” is the totalof myristate (C_(14:0)), palmitate (C_(16:0)), stearate (C_(18:0)),arachidate (C_(20:0)), behenate (C_(22:0)) and lignocerate (C_(24:0)).Fatty acid profiles reported herein were obtained by GC (using AOCSMethod Ce 1e-91).

[0028] As used herein, a “variety” is a group of plants that displaylittle or no genetic variation between individuals for at least onetrait. Varieties may be created by, e.g., several generations ofself-pollination and selection, or vegetative propagation from a singleparent using tissue or cell culture techniques.

[0029] As used herein, a “line” refers to a plant and its progenyproduced from a single transformation.

[0030] Nucleic Acid Constructs

[0031] A nucleic acid construct useful in the invention comprises amulti-functional cytosolic acetyl coA-carboxylase (ACCase) codingsequence operably linked to a promoter. Suitable cytosolic ACCasesinclude plant and animal cytosolic ACCases from organisms such asArabidopsis thaliana (e.g., GenBank Accession No. L27074), Brassicanapus (e.g., GenBank Accession No. X77576), Zea mays (e.g., GenBankAccession No. A25273) and Homo sapiens (e.g., GenBank Accession No.U19822). For example, a construct can contain a 35S cauliflower mosaicvirus (CaMV) promoter and an alfalfa (i.e., Medicago sativa) cytosolicACCase cDNA (e.g., GenBank Accession No. L25042).

[0032] Alternatively, a construct of the invention can contain ACCasenucleic acid sequences from Saccharomyces cerivisiae (e.g., GenBankAccession No. M92156), Schizosaccharomycespombe (e.g. GenBank AccessionNo. D78169), Ustilago maydis (e.g., GenBank Accession No. Z46886), Bostaurus (bovine) (e.g., GenBank Accession No. AJ132890), Rattusnorvegicus (rat) (e.g., GenBank Accession No. AB004329), Ovis aries(sheep) (e.g., GenBank Accession No. X80045), Gallus gallus (chicken)(e.g., GenBank Accession No. J03541), Glycine max (soybean) (e.g.,GenBank Accession No. L42814), Avena sativa (oat) (e.g., GenBankAccession No. AF072737), Triticum aestivum (wheat) (e.g., GenBankAccession No. U39321) or Phaseolus vulgaris (bean) (e.g., GenBankAccession No. AF007803). A representative cloning strategy for producinga construct of the present invention is described herein. Other suitablemethods for engineering constructs are described elsewhere, e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory Press (1989).

[0033] As used herein, “promoter” refers to nucleic acid sequences that,when operably linked to an ACCase coding sequence, direct transcriptionof the coding sequence such that it's gene product can be produced.Promoters can be described based on their activity (e.g., constitutive,inducible, tissue-specific or temporal-specific). Constitutive promotersare generally nucleic acid sequences that direct a relatively high levelof transcription, and typically without great tissue- ortemporal-specificity. Inducible promoters are typically nucleic acidsequences that regulate transcription in response to a stimulus (e.g., aphysical or chemical stimulus). Tissue- or temporal-specific promotersare generally nucleic acid sequences that direct transcription that isbiased toward a particular tissue or time (e.g., a particulardevelopmental stage), respectively. Oftentimes, however, a promoter'sactivity does not fall under a single description.

[0034] Suitable promoters are known (e.g., Weising et al, Ann. Rev.Genetics 22:421-478 (1988)). The following are representative promoterssuitable for use in the invention described herein: regulatory sequencesfrom fatty acid desaturase genes (e.g., Brassica fad2D or fad2F, see WO00/07430); alcohol dehydrogenase promoter from corn; light induciblepromoters such as the ribulose bisphosphate carboxylase (Rubisco) smallsubunit gene promoters from a variety of species; major chlorophyll a/bbinding protein gene promoters; the 19S promoter of cauliflower mosaicvirus (CaMV); a seed-specific promoter such as a napin or cruciferinseed-specific promoter; as well as synthetic or other natural promotersthat are, for example, inducible, constitutive, tissue-specific ortemporal-specific.

[0035] A nucleic acid construct optionally may contain a nucleic acidsequence encoding a transit peptide operably linked to an ACCase codingsequence. A transit peptide facilitates transport to plastids of theACCase polypeptide to which the transit peptide is fused. Suitabletransit peptides include any transit peptide encoded by a nuclear genethat directs transport of the encoded protein into the chloroplast.

[0036] A nucleic acid encoding a cytosolic ACCase may or may not containintrons within the coding sequence. Introns are nucleic acid sequencesthat are initially transcribed into RNA and subsequently removed. Thenumber of introns in a transcript can vary, as can the size of eachintron. Introns themselves possess very little conservation, but thesplice site sequences (i.e., the sequence at the exon-intron andintron-exon junctions) typically are highly conserved among eukaryotes.In addition, introns typically possess an internal conserved sequencecorresponding to an branch site involved in intron removal. Nucleic acidsequences containing an ACCase open reading frame can be examined forintrons using, for example, software such as the Sequence AnalysisSoftware Package of the Genetics Computer Group (GCG) (University ofWisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis.53705). An ACCase nucleic acid having introns can be, for example, agenomic ACCase nucleic acid (e.g., GenBank Accession No. L27074). AnACCase nucleic acid lacking introns can be, for example, a complementaryDNA (cDNA) of an ACCase mRNA nucleic acid (e.g., SEQ ID NO:7), or can beassembled (e.g., recombinantly) from individual exonic sequences.

[0037] It should be appreciated that many different nucleic acids willencode a polypeptide having a particular cytosolic ACCase amino acidsequence. The degeneracy of the genetic code is well known in the art,i.e., many amino acids are coded for by more than one nucleotide codon.It should also be appreciated that certain amino acid substitutions canbe made within polypeptide sequences without affecting the function ofthe polypeptide. Conservative amino acid substitutions or substitutionsof similar amino acids often are tolerated without affecting polypeptidefunction. Similar amino acids can be those that are similar in sizeand/or charge properties. Similarity between amino acids has beenassessed in the art. For example, Dayhoffet al. (1978) in Atlas ofprotein Sequence and Structure, Vol. 5, Suppl. 3, pp. 345-352,incorporated herein by reference, provides frequency tables for aminoacid substitutions that can be employed as a measure of amino acidsimilarity.

[0038] Additional regulatory sequences may be useful in the nucleic acidconstructs of the present invention, including, but not limited to,polyadenylation sequences, enhancers, introns, and the like. Suchelements may not be necessary for expression of the ACCase codingsequence, although they may increase expression by affectingtranscription, stability of the mRNA, translational efficiency, or thelike. Such elements can be included in a nucleic acid construct asdesired to obtain optimal expression of the ACCase nucleic acid in thehost cell(s). Sufficient expression, however, may sometimes be obtainedwithout such additional elements. A representative reference describingcertain regulatory elements is Weising et al., Ann. Rev. Genetics22:421-478 (1988).

[0039] Transgenic Plants

[0040] In one aspect of the invention, transgenic plants are created byintroducing an ACCase nucleic acid construct into a plant cell andgrowing the plant cell into a plant. Such plants contain and express theACCase nucleic acid construct. Suitable techniques for introducingnucleic acids into plant cells to create such plants include, withoutlimitation, Agrobacterium-mediated transformation, viral vector-mediatedtransformation, electroporation and particle gun transformation.Illustrative examples of transformation techniques are disclosed in U.S.Pat. No. 5,204,253, (describing biolistic transformations), U.S. Pat.No. 6,051,756 (describing biolistic transformation of Brassica) and U.S.Pat. No. 5,188,958 (describing Agrobacterium transformation).Transformation methods utilizing the Ti and Ri plasmids of Agrobacteriumspp. typically use binary-type vectors (e.g., ptet1, pBin19)(Walkerpeach et al., in Plant Molecular Biology Manual, Gelvin &Schilperoort, eds., Kluwer Dordrecht, C1:1-19 (1994)).

[0041] Techniques are known for the introduction of DNA into dicots aswell as monocots, as are the techniques for culturing such tissues andregenerating plants. If cell or tissue cultures are used as therecipient tissue for transformation, plants can be regenerated fromtransformed cultures by techniques known to those skilled in the art.Suitable dicots include plants such as alfalfa, soybean, rapeseed (higherucic and canola), and sunflower. Monocots that have been successfullytransformed and regenerated in the art include wheat, corn, rye, rice,sorghum and asparagus (see, e.g., U.S. Pat. Nos. 5,484,956 and5,550,318).

[0042] Preferred species for generating transgenic plants of the presentinvention include, without limitation, oil-producing species, such assoybean (Glycine max), rapeseed (e.g., Brassica napus, B. rapa and B.juncea) (both Spring and Winter maturing types within each species),sunflower (Helianthus annus), castor bean (Ricinus communis), safflower(Carthamus tinctorius), palm (e.g., Elaeis guineensis), coconut (e.g.,Cocos nucifera), meadowfoam (e.g., Limnanthes alba alba and L.douglasii), cottonseed (e.g, Gossypium hirsutum), olive (e.g., Oleaeuropaea), peanut (e.g., Arachis hypogaea), flax (e.g., Linumusitatissimum), sesame (e.g., Sesamum indicum) and crambe (e.g., Crambeabyssinica or C. hispanica). Accordingly, suitable families include, butare not limited to, Solanaceae, Leguminaceae, Brassicaceae andAsteraceae. A transgenic plant of the invention typically is a member ofa plant variety within the families or species mentioned above.

[0043] As used herein, a transgenic plant also refers to progeny of aninitial transgenic plant. Progeny includes descendants of a particularplant or plant variety, e.g., seeds developed on a particular plant.Progeny of a plant also includes seeds formed on F₁, F₂, F₃, andsubsequent generation plants, or seeds formed on BC₁, BC₂, BC₃, andsubsequent generation plants. Seeds produced by a transgenic plant canbe grown and then selfed (or out-crossed and selfed) to obtain plantshomozygous for the construct. Seeds can be analyzed to identify thosehomozygotes having the desired level of expression of a construct.Alternatively, transgenic plants and progeny thereof may be obtained byvegetative propagation of a transformed plant cell (for those speciesamenable to such techniques).

[0044] Transgenic plants can be used in commercial breeding programs forthe species of interest or can be crossed or bred to plants of relatedcrop species. Phenotypes conferred by expression of an ACCase nucleicacid construct can be transferred from one species to another speciesby, for example, protoplast fusion. Such breeding programs are useful toincorporate other agronomic or specialty traits of interest, e.g.,herbicide tolerance or a high oleic acid content in seeds.

[0045] Methods

[0046] In one aspect of the invention, there are provided methods ofgenerating a plant that produces seeds exhibiting a statisticallysignificant increase in oil content. The method includes introducing anucleic acid construct containing a promoter and an ACCase codingsequence into a plant and selecting progeny that produce seeds withincreased oil content as compared to seeds from a corresponding plantlacking the ACCase nucleic acid construct, e.g., seeds from a planthaving the same or similar genetic background as the transgenic plantbut which does not have the cytosolic ACCase construct. Such progeny areidentified after one or more generations of selection, e.g., onegeneration, three or more generations, or six or more generations. Byway of example, selection may be carried out initially, e.g., the firstand second generations, by selecting those progeny possessing the ACCaseconstruct, and selection in subsequent generations may be carried out byidentifying those progeny that possess the ACCase construct as well aselevated seed oil content.

[0047] Also provided by the invention are methods of producing seedswith a statistically significant increase in oil content. The methodsinclude introducing a nucleic acid construct containing a promoter andan ACCase coding sequence into one or more plant cells and regeneratingsuch plant cells into one or more plants. Seeds exhibiting statisticallysignificantly increased oil content can then be harvested from selectedprogeny of the plant.

[0048] Further provided by the invention are methods of increasing theoil content in seeds. The methods include introducing a nucleic acidconstruct containing a promoter and a cytosolic ACCase coding sequenceinto a plant and selecting progeny after at least one generation ofselection that produce seed with increased oil content as compared tocorresponding seeds produced from plants lacking the recombinant ACCasenucleic acid.

[0049] The following Table provides relative percent oil and proteincontent on a dry weight basis (unless indicated otherwise) in severalplants, particularly oilseed plants, that can be used in the presentinvention. Plant % Oil % Protein Key Soybean ˜20 ˜40 c (Glycine max)Rapeseed 40-44 38-41 c; d (Brassica napus) (oil free meal) Sunflower 40d (Helianthus annus) Castor bean 50 a (Ricinus communis) Safflower36.8-47.7 15.4-22.5 d (Carthamus tinctorius) Crambe 30-35 ˜28 b (Crambeabyssinica) Palm 20 c; per fresh fruit bunch (Elaeis guineensis) >50(˜20% moisture); dried kernels Coconut 34 3.5 d; coconut flesh (50%(Cocos nucifera) 69 moisture); dried kernels Maize 3.1-5.7 6-12 c; d(Zea mays) Cottonseed 25-30 25-30 d; kernel (Gossypium hirsutum) Olive19.6 1.6 fruit (52.4% moisture) (Olea europaea) Peanut 36-56 25-30 c;(unknown moisture) (Arachis hypogaea) Flax 35-45 d; per fruit capsule(Linum usitatissimum) (˜10 seeds/fruit) Sesame 53.3-57.5 25-30 d; (5-7%moisture) (Sesamum indicum)

[0050] The present invention describes a novel method of making plantsthat produce seeds with a statistically significant increase in oilcontent. As used herein, “statistically significant” refers to a p-valueof less than 0.05, e.g., a p-value of less than 0.025 or a p-value ofless than 0.01, using an appropriate measure of statisticalsignificance, e.g., a one-tailed two sample t-test.

[0051] Plants of the invention produce seeds that exhibit an increase inoil content that is statistically significant relative to seeds producedby plants that lack a cytosolic ACCase construct. Plants produced by themethod of the present invention produce seeds having an increase in oilof from about 5% to about 25% over the oil content in seeds produced byuntransformed control plants. For example, the increase in oil contentfor plants described herein is from about 5% to about 20%, or from about:5% to about 15%, or from about 10% to about 20%, relative to plantsthat lack a cytosolic ACCase construct.

[0052] The seeds of several different Brassica napus lines from a−7ACCase/Westar transformation have been deposited with the AmericanType Culture Collection (ATCC), 10801 University Blvd., Manassas, Va.,20110-2209, and have the following accession numbers. Line Accession No.Deposit Date Tao-001-30-02 Tao-001-31-02 Tao-001-56-01 Tao-001-56-06Tao-001-65-08

[0053] Seeds and plants of plant varieties made from the transgenicplants described herein are included within the scope of the invention,as well as progeny of these varieties that possess the novelcharacteristics recited herein. Oil extracted from such varieties orfrom similar varieties is also within the scope of the invention.

[0054] Nucleic acid constructs, plants and methods described hereinprovide for more efficient production of oil for food and industrialapplications (e.g., engine lubricants, hydraulic fluids, etc.). Forexample, plants described herein produce a greater yield of oil per acreplanted compared to plants lacking a cytosolic ACCase construct. Inaddition, there is increased oil yield during the processing of suchseeds.

[0055] The invention will be further described in the followingexamples, which do not limit the scope of the invention described in theclaims.

EXAMPLES Example 1 Constructs

[0056] The pSP72 vector (Promega) was digested with XhoI and SalI andsubsequently religated to remove the PvuII site. This modified vectorwas designated ModpSP72. The tobacco small subunit Rubisco (ssRubisco,also known as ribulose 1,5-bisphosphate carboxylase) transit peptide wasamplified by PCR from a tobacco ssRubisco gene/pet11d template using a5′ primer (5′-CAUCAUCAUCAUATCGATAGGTACCAAAAAAAA CAACCATGGCTTCCTCAGTTCTT)(SEQ ID NO:1) and a 3′ primer (5′-CUACUAC UACUAGCTAGCCATGGACTTCTTGTTAATTGGTGGCCA) (SEQ ID NO:2). The 5′ primer was designed tocontain ClaI, KpnI, and NcoI sites, and the 3′ primer was engineered tocontain NcoI and NheI sites. The amplified transit peptide DNA wasannealed into the pAMP1 vector (Gibco BRL) and both strands weresequenced to confirm fidelity. This construct was designated+Transit/pAMP1. To generate a construct lacking the transit peptide(−Transit/pAMP), +Transit/pAMP was digested with NcoI and religated.

[0057] The cloning of a fragment designated 209/180 from an alfalfacytosolic acetyl coA-carboxylase (ACCase) into the pAMP1 vector toproduce 209/180/pAMP1 is described in Shorrosh et al. (1994, Proc. Natl.Acad. Sci. USA, 91:4323-27). A fragment designated 147/136 was PCRamplified using primers 147 and 136 (Shorrosh et al., 1994), which wassubsequently subcloned into the pAMP1 vector to generate a 147/136/pAMP1construct. The 209/180 fragment was removed from the pAMP1 vector bydigesting with KpnI and BamHI and subcloned into the KpnI/BamHI sites ofModpSP72 to generate a 209/180/pSP72 construct. The 147/136/pAMP1construct was digested with SnaBI and BamHI and the insert containingthe 147/136 fragment was subcloned into the SnaBI/BamHI sites of the209/180/pSP72 construct to generate a 209/136/pSP72 construct.

[0058] Clone “T1”, corresponding to a partial alfalfa ACCase cDNA anddescribed in Shorrosh et al., 1994, was digested with PvuII and BamHIand subcloned into the 209/136/pSP72 construct at the PvuII/BamHI sitesto generate 209-T/pSP72. Additionally, a clone designated 3X,corresponding to a partial alfalfa ACCase cDNA (essentially the M2fragment as described in Shorrosh et al., 1994, with additional 5′ and3′ flanking sequences to facilitate cloning), was digested withEcoR47III and BamHI and subcloned into the 209-T/pSP72 construct at theEcoR47III/BamHI sites to generate 209-3X/pSP72. This construct containsa full-length alfalfa cytosolic ACCase cDNA coding sequence in the pSP72vector.

[0059] The 209-3X/pSP72 construct was digested with KpnI and BamHI andsubcloned into the +Transit/pAMP1 construct at the KpnI/BamHI sites togenerate a construct designated +6ACCase/pAMP 1. +6ACCase/pAMP1 containsa full-length alfalfa ACCase cDNA with a transit peptide at the 5′ endin the same reading frame as the ACCase coding sequence. The+6ACCase/pAMP construct was then digested with NheI and BamHI and thefull-length alfalfa ACCase cDNA, including the transit peptide, wassubcloned into the Agrobacterium binary vector, ptet1 (provided by Dr.C. Gatz, Institute fur Genbiologische, Berlin), at the NheI/BamHI sitesadjacent to the cauliflower mosaic virus (CaMV) 35S promoter. Thismanipulation generated +6ACCase/ptet1. Similarly, the 209-3X/pSP72construct was digested with Nhe/BamHI and the full-length alfalfa ACCasecDNA was subcloned into the −Transit/pAMP1 construct at the NheI/BamHIsites to generate −7ACCase/pAMP1. The −7ACCase/pAMP1 construct was thendigested with KpnI and BamHI and the full-length alfalfa ACCase cDNA wassubcloned into the ptet1 binary vector at the KpnI/BamHI sites adjacentto the CaMV 35S promoter to produce −7ACCase/ptet1. The -7ACCase/ptet1construct contains a full-length alfalfa ACCase cDNA but lacks a transitpeptide.

Example 2 Transgenic Plants

[0060] The +6ACCase/ptet1 and −7ACCase/ptet1 constructs of Example 1were used to transform Agrobacterium LBA4404. The resultingAgrobacterium transformants were each co-cultivated separately with B.napus hypocotyls and cultured consecutively on incubation, selection(containing kanamycin) and regeneration media until green shoots wereproduced. Regenerated plantlets were transferred to the greenhouse andgrown to maturity. Each T1 plant (N=240) was selfed and the resulting T2seeds were harvested from each individual T1 plant.

[0061] The ACCase constructs were introduced into B. napus hypocotyls ofthree different canola varieties as follows. A construct designated−7ACCase was introduced into Westar, a canola variety registered inCanada; and a construct designated +6ACCase was introduced into Oscar, acanola variety registered in Australia (App. No.1992/009, 19 Jun., 1996)or IMC 03, a Cargill proprietary low linolenic acid canola variety.Table 1 shows a typical fatty acid profile for each of the Brassicavarieties used in the transformations. TABLE 1 Typical fatty acidprofile of Westar, Oscar and IMC 03 seeds Fatty acid Westar Oscar IMC 03C_(16:0) 3.7¹ 3.7 3.9 C_(16:1) 0.1 0.1 0.1 C_(18:0) 2.5 2.5 1.95C_(18:1) 65.0 60 65.6 C_(18:2) 17.6 22.0 18.0 C_(18:3) 8.0 10.0 3.00C_(20:0) 0.5 0.5 0.5 C_(20:1) 1.3 1.3 1.5 C_(20:2) 0.1 0.1 0.1 C_(22:0)0.1 0.1 0.1 C_(22:1) 0.1 0.1 0.05 C_(24:0) 0.1 0.1 0.1 C_(24:1) 0.1 0.10.1 FDA 6.9 6.9 6.55 % Oil 45.0 41.0 46.0 % Protein 26.97^(2,3) — 22.46³

Example 3 Preparation of Fatty Acid Methyl Esters and Fatty AcidAnalysis of Seed by Capillary Gas Liquid Chromatography (GLC)

[0062] The following describes a means for quantifying fatty acidcomposition in canola seed. To prepare samples, approximately 150 mg ofseed is placed into a 15 ml polypropylene centrifuge tube. The seed isbroken apart and 0.6 ml of methanolic KOH solution is added to the tube.After mixing on a vortex mixer for approximately 30 sec, the tube isplaced in a water bath at 60° C. for 60 sec. About 4.0 ml of saturatedNaCl solution is added to the tube followed by 1.0 ml of iso-octane andthe sample mixed on a vortex mixer for an additional 30 sec. The sampleis centrifuged for 5 min to separate and purify the organic layer.Approximately 700 μl of the organic layer, which contains the fatty acidmethyl esters, is removed from the tube and placed into a GC autosamplervial. The vial is purged with nitrogen gas to remove the oxygen andpreserve the sample.

[0063] Samples are analyzed, based on AOCS Method Ce 1e-91, by injecting1.0 μl into a Hewlett Packard 6890 gas chromatograph by means of anautosampler. A normalized percentage is calculated and reported for eachfatty acid in the sample.

[0064] The GC conditions are as follows:

[0065] Column: 5 m×0.32 mm DB-Wax (0.5 μm film thickness);

[0066] Detector: FID;

[0067] Inlet temp.: 250° C.;

[0068] Detector temp.: 250° C.;

[0069] Split ratio: 100:1;

[0070] Carrier gas: helium at 30.0 ml/min; and

[0071] Oven program: 1.0 min at 220° C.; 10° C./min up to 245° C.; and3.0 min at 245° C.

Example 4 Determination of Oil and Moisture Content in Canola Seed byNMR Spectroscopy

[0072] The following is a non-destructive method for determining oil andmoisture content in samples of canola seed by means of nuclear magneticresonance spectroscopy. An Oxford MQA6005 NMR Analyzer (OxfordAnalytical Instruments Limited, Concord, Mass.) is calibrated accordingto manufacturer specifications. Six samples of canola seed (˜22g/sample) are used for calibration. Samples are selected to representthe oil and moisture ranges over which most unknown samples are expectedto fall. The oil content of each sample is determined by Soxhletextraction (based on AOCS Method Am 2-93). Moisture content isdetermined by gravimetric means (based on AOCS Method Ai 2-75). Theresponse of each sample is then measured on the NMR instrument. Twocalibration curves (one for oil and one for moisture) are prepared usingthe data collected.

[0073] Samples containing unknown amounts of oil and moisture areanalyzed according to the instrument manufacturer instructions (based onAOCS Recommended Practice Ak 4-95). The response of each sample iscollected and stored by a computer. The results are calculated andexpressed as “Oil %”, “Moisture %”, and “Oil % Normalized to Dry Mass”(conversion from Oil % (as is) to Oil % on a dry basis is calculatedusing the following formula: Oil % (dry) =Oil % (as is)/[1-(Moisture%/100)]).

Example 5 Determination of Percent Oil, Moisture, Protein, Chlorophyll,and Fatty Acids by NIR Spectroscopy

[0074] The following method provides a means of predicting the levels ofoil, moisture, protein, chlorophyll, oleic acid (C_(18:0)), linoleicacid (C_(18:1)), and linolenic acid (C_(18:2)) in canola seed samples bymeans of near infra-red reflectance spectroscopy.

[0075] A Foss NIR Systems model 6500 Feed and Forage Analyzer (FossNorth America, Eden Prairie, Minn.) is calibrated according to themanufacturer's recommendatiors. Canola seed samples, which representedwide ranges of the sample constituents listed above, are collected forcalibration. Lab analysis results are determined using acceptedmethodology (i.e., oil, AOCS Method Ak 3-94; moisture, AOCS Method Ai2-75; fatty acid, AOCS Method CE 1e-91 and AOCS Method CE 2-66;chlorophyll, AOCS Method CC 13D-55; protein, AOCS Method BA 4e-93; andglucosinolates, AOCS Method Ak 1-92). Instrument response is alsomeasured for each sample. A calibration equation is calculated for eachconstituent by means of chemometrics. These equations are combined intoone computer file and are used for prediction of the constituentscontained in unknown canola samples.

[0076] Seed samples containing unknown levels of the above constituentsare prepared by removing foreign material from the sample. Cleaned wholeseed is placed into the instrument sample cell and the cell is placedinto the instrument sample assembly. Analysis is carried out accordingto instrument manufacturer instructions (based on AOCS Procedure Am1-92). The results are predicted and reported as % constituent (% oiland protein are reported based on dry weight). Conversion from ‘dryweight’ basis to ‘as is’ basis for oil and protein is calculated usingthe following formula:

constituent (as is)=constituent (dry wt.)×[1−(% moisture/100)].

Example 6 T1 plants and T2 seeds

[0077] A total of 126−7ACCase/Westar plants were regenerated in agreenhouse from the plantlets described in Example 2. Each T1 plant wasselfed and a sample of T2 seeds from each plant was analyzed for fattyacid composition by gas chromatography as described in Example 3. T2seeds had fatty acid compositions that were not significantly differentfrom the fatty acid profile of the Westar background variety.

[0078] Table 2 shows the mean fatty acid profile (±standard deviation)for the −7ACCase/Westar transformation. T2 seeds from each T1 plant wereadvanced (i.e., no selection was performed on T2 seeds) such that 5-10seeds from each T1 plant were grown individually in a single row in thegreenhouse. TABLE 2 Mean fatty acid profile of T2 seeds Fatty acid-7ACCase/Westar C_(14:0)  0.07 (0.04)¹ C_(16:0) 4.59 (0.69) C_(16:1)0.31 (0.13) C_(18:0) 2.28 (0.45) C_(18:1) 61.61 (3.53)  C_(18:2) 20.18(2.27)  C_(18:3) 7.95 (1.04) C_(20:0) 0.77 (0.12) C_(20:1) 1.23 (0.10)C_(20:2) 0.08 (0.02) C_(22:0) 0.45 (0.09) C_(22:1) 0.03 (0.03) C_(24:0)0.24 (0.10) C_(24:1) 0.24 (0.17) FDA 8.39 (1.22)

[0079] Table 3 shows fatty acid profiles of T2 seeds from representativeindividual lines from the −7ACCase/Westar transformation. TABLE 3 Fattyacid profile of T2 seeds from representative individual -7ACCase/WestarBrassica lines 001-01¹ 001-120 001-121 001-31 C_(14:0) 0.103² 0.4480.154 0.056 C_(16:0) 7.720 7.053 6.339 4.222 C_(16:1) 0.776 0.821 0.8200.249 C_(18:0) 3.181 3.107 4.213 1.988 C_(18:1) 55.584 51.827 50.62464.292 C_(18:2) 22.659 24.794 25.949 18.319 C_(18:3) 6.806 8.343 7.0418.106 C_(20:0) 1.170 0.969 1.235 0.688 C_(20:1) 0.950 0.815 0.902 1.337C_(20:2) 0.000 0.065 0.082 0.065 C_(22:0) 1.170 0.578 0.912 0.381C_(22:1) 0.000 0.024 0.188 0.000 C_(24:0) 0.379 0.421 0.574 0.168C_(24:1) 0.000 0.735 0.966 0.129 FDA 13.225 12.576 13.427 7.504

Example 7 T2 Plants and T3 Seeds

[0080] A total of 834 T2 plants from 126 lines were selfed and theresulting T3 seed analyzed for fatty acid composition by gaschromatography as described in Example 3.

[0081] Table 4 shows summary statistics (mean±standard deviation) offatty acid profiles of seeds from the total population of T3 plantsproduced in the −7ACCase/Westar transformation, from those T3 plantsselected for advancement and from plants corresponding to thenon-transgenic Westar variety. Data for the non-transgenic controlplants was obtained from 19 Westar plants grown under similarconditions. TABLE 4 Mean fatty acid profile of T3 seeds Fatty-7ACCase/Westar acid Total¹ Sel¹ Control¹ C_(14:0) 0.06² 0.06 0.06(0.02) (0.03) (0.02) C_(16:0) 4.71 4.90 4.45 (0.79) (1.03) (0.51)C_(16:1) 0.27 0.28 0.23 (0.10) (0.13) (0.10) C_(18:0) 2.84 3.25 2.68(0.68) (0.68) (0.52) C_(18:1) 66.62 67.20 69.76 (5.34) (6.69) (3.41)C_(18:2) 15.67 14.46 13.78 (3.50) (3.88) (2.46) C_(18:3) 5.68 5.20 4.90(1.43) (1.26) (1.05) C_(20:0) 1.14 1.32 1.08 (0.26) (0.21) (0.29)C_(20:1) 1.38 1.43 1.41 (0.21) (0.24) (0.14) C_(20:2) 0.07 0.06 0.05(0.02) (0.02) (0.02) C_(22:0) 0.74 0.87 0.75 (0.22) (0.21) (0.16)C_(22:1) 0.03 0.03 0.02 (0.04) (0.04) (0.02) C_(24:0) 0.50 0.60 0.55(0.16) (0.13) (0.15) C_(24:1) 0.31 0.34 0.28 (0.13) (0.13) (0.06) FDA9.99 11.00 9.56 (1.72) (1.84) (1.09)

[0082] Three hundred ninety-five plots of T2 plants (representing 104lines) from the −7ACCase/Westar transformation were selected foradvancement based on T3 seeds exhibiting one or more of the followingproperties in fatty acid composition: C_(18:0)>3.45%, C_(18:2)<13.1%,C_(18:3)<4.51%, C_(20:0)>1.55%, or FDA saturates (defined as the sumC_(16:0), C_(18:0), C_(20:0), C_(22:0) and C_(24:0))>10.5%.

[0083] Table 5 shows the fatty acid profile of T3 seed fromrepresentative individual lines from the −7ACCase/Westar transformationthat were selected for advancement. Bolded numbers indicate criteriaused to select and advance the plants. TABLE 5 Fatty acid profile of T3seeds from representative -7ACCase/Westar lines selected for advancementFatty 001-26- 001-27- 001-30- 001-31- 001-31- 001-78- acid 01¹ 12 02 0507 04 C_(14:0) 0.0755² 0.1100 0.0371 0.0393 0.0481 0.1419 C_(16:0)7.3213 10.6772 3.9496 3.8797 4.1826 10.7381 C_(16:1) 0.3434 0.62300.1360 0.1408 0.2025 0.6661 C_(18:0) 4.1630 2.9979 2.1986 2.6323 3.03417.5048 C_(18:1) 42.6632 28.1662 72.7333 72.5224 70.0784 31.4668 C_(18:2)29.2415 37.0096 11.5253 11.4008 12.2550 29.4236 C_(18:3) 9.5494 12.47354.5227 4.6344 5.1038 10.4718 C_(20:0) 1.7108 1.5811 1.1132 1.2070 1.38212.6041 C_(20:1) 2.2521 2.4504 1.7807 1.6792 1.6406 2.6668 C_(20:2)0.1620 0.1600 0.0726 0.0724 0.0700 0.1295 C_(22:0) 1.1188 1.7861 0.95340.9449 0.9628 1.8995 C_(22:1) 0.1219 0.2030 0.1075 0.0875 0.0000 0.0000C_(24:0) 0.7574 0.9286 0.5629 0.5129 0.6498 1.6442 C_(24:1) 0.51970.8334 0.3071 0.2465 0.3903 0.6428 FDA 15.1468 18.0809 8.8148 9.216110.2594 24.5325

Example 8 T3 Plants and T4 Seeds

[0084] About 0.5 g of T3 seed from each T2 plant selected foradvancement as described in Example 7, were planted in field plots inColorado, USA. T4 seeds were collected and combined from 20 random T3plants from each line and analyzed for fatty acid composition (by G C;see Example 3) and oil content (by NMR; see Example 4). Following randombulk T4 seed analysis from each plot, 5-10 T4 seeds from those linesexhibiting increased oil content were advanced individually in thegreenhouse.

[0085] Thirteen T3 lines with oil content of 48.7% to 50% were advancedand one T3 line with oil content of 48.1% was advanced from the−7ACCase/Westar transformation. Table 6 shows summary statistics(mean±standard deviation) for seed fatty acid profiles of the total T4population, the plants selected for advancement and correspondingnon-transgenic control plants. Data for the non-transgenic controlpopulation was obtained from 139 Westar plants transgenic for an fae1gene. The fae1 gene elongates C_(18:1) to C_(20:1), thereby resulting inan accumulation of C_(20:1) in plants transgenic for fae1, but does notaffect oil content. TABLE 6 Mean fatty acid profile and oil content ofT4 seeds Fatty -7ACCase/Westar acid Total¹ Sel¹ Control¹ C_(14:0) 0.06²0.06 0.07 (0.01) (0.01) (0.03) C_(16:0) 3.52 3.52 3.48 (0.40) (0.12)(0.52) C_(16:1) 0.19 0.18 0.19 (0.03) (0.01) (0.05) C_(18.0) 3.00 2.122.15 (8.76) (0.13) (0.24) C_(18:1) 68.58 69.83 64.60 (6.64) (0.33)(8.61) C_(18:2) 15.26 15.30 14.68 (1.98) (0.44) (1.32) C_(18:3) 6.426.40 6.49 (0.90) (0.29) (0.58) C_(20:0) 0.71 0.66 0.75 (0.11) (0.01)(0.18) C_(20:1) 1.26 1.18 5.47 (0.19) (0.05) (7.83) C_(20:2) 0.05 0.050.14 (0.01) (0.00) (0.18) C_(22:0) 0.34 0.29 0.32 (0.04) (0.01) (0.04)C_(22:1) 0.08 0.05 0.92 (0.44) (0.08) (2.23) C_(24:0) 0.30 0.22 0.22(0.29) (0.04) (0.09) C_(24:1) 0.24 0.15 0.52 (0.25) (0.09) (0.57) FDA7.92 6.86 6.98 (8.58) (0.16) (0.65) % Oil 45.7 49.1 45.5 (3.2) (0.51)(1.92)

[0086] Table 7 shows the fatty acid profiles of T4 seed fromrepresentative individual lines from the −7ACCase/Westar transformationthat were selected for advancement. TABLE 7 Fatty acid profile and oilcontent of T4 seeds from representative -7ACCase/Westar lines selectedfor advancement Fatty 001-31- 001-30- 001-31- 001-30- acid 07¹ 02 06 05C_(14:0) 0.06² 0.06 0.06 0.07 C_(16:0) 3.59 3.52 3.42 3.70 C_(16:1) 0.190.17 0.17 0.18 C_(18:0) 2.01 2.10 2.07 2.10 C_(18:1) 69.76 69.47 70.1269.49 C_(18:2) 15.64 16.21 15.08 15.49 C_(18:3) 6.24 5.99 6.48 6.45C_(20:0) 0.65 0.65 0.66 0.67 C_(20:1) 1.13 1.14 1.18 1.15 C_(20:2) 0.040.05 0.05 0.05 C_(22:0) 0.30 0.28 0.30 0.29 C_(22:1) 0.02 0.02 0.02 0.01C_(24:0) 0.23 0.23 0.26 0.23 C_(24:1) 0.14 0.12 0.14 0.12 FDA 6.84 6.846.76 7.06 % Oil 50.0 50.0 49.4 49.4

Example 9 T4 Plants and T5 Seeds

[0087] T4 seeds from 10 random selfed plants representing each lineselected for advancement in Example 8 were planted in a greenhouse using5-10 seeds per row. T4 plants were selfed, and T5 seeds were collectedfrom individual plants. A portion of the T5 seeds from each line werecombined and analyzed for oil content and fatty acid analysis by NIR asdescribed in Example 5.

[0088] Table 8 shows summary statistics (mean±standard deviation) forseed oil and seed protein content for the total T5 population, for T5lines selected for advancement and for corresponding non-transgeniccontrols. Data for the Westar control plants was obtained from ‘controlsamples’. Each ‘control sample’ contained seed bulked from approximately20 control plants.

[0089] Forty-five T4 plants (representing 6 lines from 14 plots) fromthe −7ACCase/Westar transformation yielded seed having an oil content of44.4% to 50.4% and 7 of those plants, representing 2 lines (001-30-02and 001-31-07) yielding seed having an oil content ranging from 44.4% to50.1%, were advanced. TABLE 8 Oil content, protein content and fattyacid profile of T5 seeds Fatty -7ACCase/Westar acid Total¹ Sel¹ Control¹C_(18:1) 69.00² 69.40 68.15 (0.90) (0.60) (0.56) C_(18:2) 13.60 13.2014.60 (1.30) (29.00) (0.68) C_(18.3) 7.60 7.80 7.09 (0.60) (0.30) (0.50)% Oil 49.90 49.90 45.64 (1.60) (1.60) (1.22) % 20.00 20.50 24.20 Protein(1.10) (1.20) (0.22) Chlor³ 36.60 37.10 18.15 (18.20) (25.60) (4.86)Gluc⁴ 4.80 4.60 ND (1.00) (1.10) ND

[0090] Table 9 shows the fatty acid profiles of T5 seed fromrepresentative individual lines from the −7ACCase/Westar transformationthat were selected for advancement. TABLE 9 Oil content, protein contentand fatty acid profile of T5 seed from representative -7ACCase/Westarlines selected for advancement 001-30- 001-30- 001-30- 001-31- 02¹ 02 0207 C_(18:1) 69.0² 69.6 70.4 69.2 C_(18:2) 12.6 12.5 12.3 14.1 C_(18:3)8.5 8.1 7.6 7.8 % Oil 50.3 50.2 49.8 52.3 % Protein 20.9 20.3 20.6 18.4Chlor³ 63.9 20.8 31.3 13.7 Gluc⁴ 5.8 4.1 4.2 3.2

Example 10 T5 Plants and T6 Seeds

[0091] T5 lines that were selected based on percent oil and proteincontent as described in Example 9 were advanced in the field inColorado, USA and in Saskatchewan, Canada. Approximately 0.5 g of seedsfrom each selected line were planted and selfed. At maturity, T6 seedswere collected from 20 plants of each line and pooled for analysis ofoil content and fatty acid composition by NIR. Based upon NIR analysisand favorable oil content in the pooled sample of T6 seed, T6 seed from10 random T5 plants from each line were advanced in the greenhouse.

[0092] Two lines from the −7ACCase/Westar transformation from T5 plantsgrown in Canada had T6 seeds that exhibited an oil content of 38.4% to49.5%. The protein content in seeds harvested from the Canada-grownplants was measured in air-dried, oil-free seed meal (using the ‘GenericCombustion Method for Determination of Crude Protein’, AOCS Method Ba4e-93), and the mean was determined to be 46.62% (±1.33) in T6 seed fromthe −7ACCase/Westar transformation. Percent protein content as shown inTable 10 for the Canadian samples was estimated based upon the percentprotein content reported for the air-dried, oil-free seed meal using theoil content reported and assuming a moisture content of 5%.

[0093] T5 plants representing six lines of the −7ACCase/Westartransformation grown in Colorado, USA produced seed that had an oilcontent of 41.9% to 51.0%, and plants from five different lines, havingan oil content of 48.8% to 50.5%, were advanced.

[0094] Table 10 shows the mean oil content (±standard deviation) of theT6 plants and control plants grown in Canada, and Table 11 shows thecorresponding data for the total population of T6 plants grown in theUSA, those T6 plants selected for advancement and from non-transgeniccontrol plants grown in the USA. USA-grown controls for the−7ACCase/Westar transformation consisted of 2 control samples each ofIMC129 and IMC130 (IMC 129 and IMC130 are both related in the followingway to the Westar variety: IMC129 carries a mutation and is otherwise≧99% Westar background, while IMC130 is the result of a cross betweenIMC01 and IMC129 varieties, and therefore, contains ≦50% of the Westarbackground). Canadian-grown controls for the −7ACCase/Westertransformation consisted of 2 IMC130 control samples. TABLE 10 Oilcontent, protein content and fatty acid profile of Canadian-grown T6seeds -7ACCase/Westar Total¹ Control¹ % Oil 46.78² 42.69 (2.88) (0.18) %Protein 26.12³ 26.66³

[0095] TABLE 11 Oil content, protein content and fatty acid profile ofUSA-grown T6 seeds -7ACCase/Westar Total¹ Sel¹ Control¹ C_(18:1) 68.71²68.54 68.19 (1.64) (0.77) (6.76) C_(18:2) 15.17 15.09 16.03 (1.26)(0.78) (5.12) C_(18:3) 8.05 8.02 7.44 (0.56) (0.23) (2.36) % Oil 47.8449.48 48.54 (1.45) (0.66) (1.65) % Protein 25.18 23.45 23.84 (1.45)(1.09) (1.90) Chloro³ −3.59 −5.67 −0.77 (2.99) (1.02 (3.35) Gluc⁴ 5.234.87 5.35 (0.64) (0.69) (1.24)

[0096] Using a one-tailed, two-sample Student's t-test, results from theT6 seeds were evaluated for statistical significance. The average oilcontent of the total T6 population from Canadian field plots wascompared with the average oil content from the correspondingnon-transgenic plants grown in Canada, while the T6 population selectedfor advancement from field plots in the USA was compared with thecorresponding USA-grown control population for each line.

[0097] The −7ACCase/Westar plants grown in Canada (n=35) had an averageoil content that was significantly higher (p<0.1) than that of thecontrol population (n=2).

[0098] The −7ACCase/Westar plants grown in the USA and selected foradvancement (n=17) had a higher oil content that was statisticallysignificant (p<0.05) compared to the average oil content of the controlplants (n=5).

[0099] Table 12 shows the percent oil content in T6 seeds from 6representative individual lines selected for advancement from the−7ACCase/Westar transformation. Control plants grown in the field inColorado, USA produced seeds that exhibited an average oil content of44.62 (on a dry weight basis). TABLE 12 Oil content of USA-grown T6seeds from individual -7ACCase/Westar lines selected for advancementLine % Oil Tao-001-30-02 48.86-49.58 Tao-001-31-02 49.93-50.51Tao-001-56-01 48.86-49.42 Tao-001-56-06 48.84-50.39 Tao-001-65-0849.10-49.85

Example 11 PCR Analysis

[0100] A nickel size portion of leaf tissue was taken at 2.5 weekspost-germination from 12 T7 plants (representing 12 different−7ACCase/Westar transformed lines) grown from the T6 seeds described inExample 10. Tissue samples were dried in a food dehydrator at 135° C.for 8-16 hrs. DNA was isolated using the Qiagen Dneasy 96 Plant Kit andresuspended in 150 μl buffer.

[0101] PCR amplification was performed in a volume of 20 μl containingthe following: 1×PCR Buffer containing 1.5 mM MgCl₂ (Qiagen PCR CoreKit); 0.2 mM DNTP; 0.5 units Taq polymerase (Qiagen); 0.5 μM MF-ACCase119 primer (5′-GTAGGCACCCTGCTACTACA (SEQ ID NO:9)); 0.5 μM MF-ACCase 645primer (5′-CATCAGGAATAGTAATCAAGTCA (SEQ ID NO:10)); 0.4% sucrose; 0.008%Cresol. A 30 cycle amplification was performed using the following PCRconditions: denaturation at 94° C. for 30 sec, annealing at 55° C. for30 sec, and extension at 72° C. for 60 sec. PCR products were analyzedby 1.2% agarose gel electrophoresis and visualized by ethidium bromidestaining. PCR products of the predicted size were detected in all 12−7ACCase/Westar plants analyzed, indicating the presence of the alfalfacytosolic ACCase gene in all lines examined.

Example 12 Crosses Between the T6 Plants

[0102] T6 seeds from the selected lines shown in Table 12 were grown inthe field in Colorado, USA. Reciprocal crosses were made between the T6plants derived from the −7ACCase/Westar transformation (lacking atransit peptide) and two T6 plants derived from a +6ACCase/IMC 03transformation (having a transit peptide). Plants were grown to maturityand the seeds were harvested. F1 seeds are grown and the resultingplants are allowed to self-pollinate. The resulting F2 progeny seeds areharvested, and the fatty acid profile is determined and oil and proteincontent are analyzed as described in Examples 3-5. Samples exhibiting astatistically significant increase in oil content are selected foradvancement.

Example 13 Outcrosses Between the T6 Plants and Other Plant Varieties

[0103] T6 seeds from the selected lines shown in Table 12 were grown inthe field in Colorado, USA. Crosses were made between the T6 plantsderived from the −7ACCase/Westar transformation (lacking a transitpeptide) or two T6 plants derived from a +6ACCase/IMC 03 transformation(having a transit peptide) and plants of a Brassica line exhibiting higholeic acid content. An example of a high oleic acid Brassica variety isQ4275, described in PCT 96/20090. F1 seeds are grown and the resultingplants are allowed to self-pollinate. The resulting F2 progeny seeds areharvested, and the fatty acid profile is determined and oil and proteincontent are analyzed as described in Examples 3-5. Samples exhibiting astatistically significant increase in oil content, as well as high oleicacid content, are selected for advancement.

[0104] T6 seeds from the selected lines shown in Table 12 were grownin-the field in Colorado, USA. Crosses were made between the T6 plantsderived from the −7ACCase/Westar transformation (lacking a transitpeptide) or two T6 plants derived from a +6ACCase/IMC 03 transformation(having a transit peptide) and plants of a Brassica line exhibitingelevated oil content but lacking an ACCase construct. Examples ofBrassica varieties exhibiting elevated oil are IMC106RR and IMC107RR,proprietary Cargill Brassica lines. The oil content in IMC106RR orIMC107RR is about 46.5-47% and 47.5-48%, respectively, on a dry weightbasis. Another example of a Brassica line that exhibits elevated oilcontent is Polo, a non-transgenic variety registered in Canada(Registration # AGO12). Polo has an oil content of about 48.5-49.5% on adry weight basis. F1 seeds were grown in the greenhouse and theresulting plants allowed to self-pollinate. The resulting F2 progenyseeds are harvested, and the fatty acid profile is determined and oiland protein content are analyzed as described in Examples 3-5. Seedsexhibiting an oil content that is significantly higher than eitherparental line are selected for advancement. Progeny plants are allowedto self-pollinate and the seeds analyzed for oil content. Those seedsexhibiting increased oil content are advanced.

[0105] T6 seeds from the selected lines shown in Table 12 were grown inthe field in Colorado, USA. Crosses were made between the T6 plantsderived from the −7ACCase/Westar transformation (lacking a transitpeptide) or two T6 plants derived from a +6ACCase/IMC 03 transformation(having a transit peptide) and plants of a Brassica line exhibiting higherucic acid content but lacking an ACCase construct. Suitable higherucic acid Brassica lines include, for example, Hero (HE101, HEC01),Mercury, Venus or Neptune which have about 45% or more erucic acid (McVetty et al., Can. J. Plant Sci., 76(2):341-342 (1996); Scarth et al.,Can. J. Plant Sci., 75(1):205-206 (1995); and Mc Vetty et al., Can J.Plant Sci., 76(2):343-344 (1996)). F1 seeds were grown in the greenhouseand the resulting plants allowed to self-pollinate. The resulting F2progeny seeds are harvested, and the fatty acid profile is determinedand oil and protein content are analyzed as described in Examples 3-5.Seeds exhibiting an oil content that is significantly higher than eitherparental line are selected for advancement. Progeny plants are allowedto self-pollinate and the seeds analyzed for oil content. Those seedsexhibiting increased oil content are advanced.

[0106] Additionally, PCR is used to examine the segregation of thealfalfa ACCase nucleic acid in the progeny of the above-describedcrosses. After crossing T6 plants derived from a −7ACCase/Westartransformation or T6 plants derived from a +6ACCase/IMC 03transformation with an appropriate plant (i.e., a plant exhibiting highoil, high oleic acid or high erucic acid), F1 seeds are harvested, grownin the greenhouse and the resulting plants are allowed toself-pollinate. The resulting F2 progeny seeds are harvested, and PCR isperformed to detect the presence of the alfalfa ACCase nucleic acidsequences using DNA extracted from the seed. Alternatively, the F2 seedsare grown into mature F3 plants, and PCR is performed, using DNAextracted from the leaves of the plant to detect the presence of thealfalfa ACCase nucleic acid sequences. Representative PCR primershomologous to the alfalfa ACCase are described in Example 11. If PCRamplification indicates the presence of the alfalfa ACCase nucleic acidsequences, oil content is then determined by NMR or NIR as described inExamples 4 and 5. Seeds or plants are subsequently advanced based upon apositive PCR amplification (i.e., the presence of the alfalfa ACCasenucleic acid sequences) and elevated oil content.

Example 14 Increased Oil Content in Crushed Seeds

[0107] T6 seeds of Example 10 are planted, allowed to pollinate, and theresulting seeds are harvested and crushed. The oil content of thecrushed seeds is about 5% to about 25% higher than the oil content in acorresponding plant lacking an ACCase construct. The oil is extractedfrom the crushed seeds as described in, e.g., U.S. Pat. Nos. 5,969,169or 5,850,026.

[0108] Briefly, the seed is cleaned through commercial seed cleaningequipment to remove foreign matter such as weed seeds, plant material,immature seed and other matter. The cleaned seed is crushed and theresulting oil is processed at the Cargill Plant (West Fargo, N.Dak.).Greater than 350 tons of seed is crushed using the processing conditionsoutlined below.

[0109] Whole seed is passed through a double roll Bauermeister flakingrolls with smooth surface rolls available from Bauermeister Inc.(Memphis, Tenn.). The roll gap is adjusted so as to produce a flake 0.25to 0.30 mm in thickness. Flaked seed is conveyed to a five-tray, 8-footdiameter stacked cooker, manufactured by Crown Iron Works (Minneapolis,Minn.). The flaked seed moisture is adjusted in the stacked cooker to5.5-6.0%. Indirect heat from the steam heated cooker trays is used toprogressively increase the seed flake temperature to 80-90° C., with aretention time of approximately 20-30 minutes. A mechanical sweep arm inthe stacked cooker is used to ensure uniform heating of the seed flakes.Heat is applied to the flakes to deactivate enzymes, facilitate furthercell rupturing, coalesce the oil droplets and agglomerate proteinparticles in order to ease the extraction process.

[0110] Heated flakes are conveyed to a screw press from AndersonInternational Corp. (Cleveland, Ohio) equipped with a suitable screwwormassembly to reduce press out of the oil from the flakes by approximately70%. The resulting press cake contains a small percentage of residualoil. Crude oil produced from the pressing operation is passed through asettling tank with a slotted wire drainage top to remove the solidsexpressed out with the oil in the screw pressing operation. Theclarified oil is passed through a plate and frame filter to remove theremaining fine solid particles. The filtered oil is combined with theoil recovered from the extraction process before oil refining.

[0111] The press cake produced from the screw pressing operation istransferred to a FOMM basket extractor available from French Oil Milland Machinery Co. (Piqua, Ohio) where the oil remaining in the cake isextracted with commercial n-hexane at 55° C. Multiple counter-currenthexane washes are used to substantially remove the remaining oil in thepress cake, resulting in a press cake that contains residual oil in theextracted cake. The oil and hexane mixture (miscella) from theextraction process is passed through a two-stage rising film tube typedistillation column to distill the hexane from the oil. Final hexaneremoval from the oil is achieved by passing the oil through a strippercolumn containing disk and doughnut internals under 23-26 in Hg vacuumand at 107-115° C. A small amount of stripping steam is used tofacilitate the hexane removal. The oil recovered from the extractionprocess is combined with the filtered oil from the screw pressingoperation, resulting in blended crude oil, and is transferred to oilprocessing.

[0112] In the oil processing, the crude oil is heated to 66° C. in abatch-refining tank, to which 0.15% food-grade phosphoric acid, as 85%phosphoric acid, is added. The acid serves to convert the non-hydratablephosphatides to a hydratable form, and to chelate minor metals that arepresent in the crude oil. The phosphatides and the metal salts areremoved from the oil along with the soapstock. After mixing for 60minutes at 66° C., the oil acid mixture is treated with sufficientsodium hydroxide solution to neutralize the free fatty acids and thephosphoric acid in the acid oil mixture. This mixture is heated to 71°C. and mixed for 35 minutes. The agitation is stopped and theneutralized free fatty acids, phosphatides, etc. (soapstock) are allowedto settle into the cone bottom of the refining tank for 6 hours. Afterthe settling period, the soapstock is drained off from the neutralizedoil.

[0113] A water wash is done to reduce the soap content of the oil byheating the oil to 82° C. and adding 12% hot water. Agitation of themixture continues for 10 minutes. The mixture is allowed to settle outfor 4 hours, at which time the water is drained off the bottom of therefining vessel. The water washed oil is heated to 104-110° C. in avacuum bleacher vessel maintained at 24-26 in. Hg vacuum. A slurry ofthe oil and Clarion 470 bleaching clay available from American ColloidCompany (Refining Chemicals Division, Arlington Heights, Ill.) is addedto the oil in the vacuum bleacher. This mixture is agitated for 20minutes before filtering to remove the bleaching clay. The clay slurryaddition is adjusted to provide a Lovibond color (AOCS Official MethodCc 136-4) of less than 1.0 red units when the oil is heated to 288° C.under atmospheric pressure. Nitrogen is injected into the filteredbleached oil and is maintained under a nitrogen blanket until the oil isdeodorized.

[0114] Refined and bleached oil is deodorized in a semi-continuousVotator deodorizer tower at a rate of approximately 7,000 pounds perhour. The deodorization temperature is maintained at 265-268° C. with asystem pressure of 0.3-0.5 mm Hg absolute pressure. Sparge steam is usedto strip off the free fatty acids, color bodies, and odor components.Retention time in the deodorizer is generally 30-90 minutes. Thedeodorized oil is cooled to 45-50° C. and nitrogen is injected prior toremoval of the vacuum. The deodorized oil is stored under a nitrogenblanket and the resulting deodorized oil analyzed for fatty acidcomposition.

Other Embodiments

[0115] It is to be understood that while the invention has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention, which is defined by the scope of the appended claims.Other aspects, advantages, and modifications are within the scope of thefollowing claims.

1 12 1 56 DNA Artificial Sequence primer for PCR 1 caucaucauc auatcgataggtaccaaaaa aaacaaccat ggcttcctca gttctt 56 2 45 DNA Artificial Sequenceprimer for PCR 2 cuacuacuac uagctagcca tggacttctt gttaattggt ggcca 45 3204 DNA Nicotiana tabacum CDS (1)...(204) 3 atg gct tcc tca gtt ctt tcctct gca gca gtt gcc acc cgc agc aat 48 Met Ala Ser Ser Val Leu Ser SerAla Ala Val Ala Thr Arg Ser Asn 1 5 10 15 gtt gct caa gct aac atg gttgca cct ttc act ggc ctt aag tca gct 96 Val Ala Gln Ala Asn Met Val AlaPro Phe Thr Gly Leu Lys Ser Ala 20 25 30 gcc tca ttc cct gtt tca agg aagcaa aac ctt gac atc act tcc att 144 Ala Ser Phe Pro Val Ser Arg Lys GlnAsn Leu Asp Ile Thr Ser Ile 35 40 45 gcc agc aac ggc gga aga gtg caa tgcatg cag gtg tgg cca cca att 192 Ala Ser Asn Gly Gly Arg Val Gln Cys MetGln Val Trp Pro Pro Ile 50 55 60 aac aag aag tcc 204 Asn Lys Lys Ser 654 68 PRT Nicotiana tabacum 4 Met Ala Ser Ser Val Leu Ser Ser Ala Ala ValAla Thr Arg Ser Asn 1 5 10 15 Val Ala Gln Ala Asn Met Val Ala Pro PheThr Gly Leu Lys Ser Ala 20 25 30 Ala Ser Phe Pro Val Ser Arg Lys Gln AsnLeu Asp Ile Thr Ser Ile 35 40 45 Ala Ser Asn Gly Gly Arg Val Gln Cys MetGln Val Trp Pro Pro Ile 50 55 60 Asn Lys Lys Ser 65 5 149 DNA ArtificialSequence representative construct (5′ end) 5 ctaacatggt tgcacctttcactggcctta agtcagctgc ctcattccct gtttcaagga 60 agcaaaacct tgacatcacttccattgcca gcaacggcgg aagagtgcaa tgcatgcagg 120 tgtggccacc aattaacaagaagtccatg 149 6 22 DNA Artificial Sequence representative construct (5′end) 6 ggtaccaaaa aaaacaacca tg 22 7 7151 DNA Medicago sativa CDS(1)...(6771) 7 atg gct agc gtg ggc cgt gga aat gga tat tta aac agt gtgcta ccg 48 Met Ala Ser Val Gly Arg Gly Asn Gly Tyr Leu Asn Ser Val LeuPro 1 5 10 15 agt agg cac cct gct act aca acc gaa gta gat gaa tac tgcaat gcc 96 Ser Arg His Pro Ala Thr Thr Thr Glu Val Asp Glu Tyr Cys AsnAla 20 25 30 ctt gga gga aac aag ccg att cat agc ata ttg att gca aac aatgga 144 Leu Gly Gly Asn Lys Pro Ile His Ser Ile Leu Ile Ala Asn Asn Gly35 40 45 atg gca gca gtc aag ttt ata cgt agt gtt agg agt tgg gct tac gag192 Met Ala Ala Val Lys Phe Ile Arg Ser Val Arg Ser Trp Ala Tyr Glu 5055 60 aca ttt ggc acg gaa aaa gct atc ttg ttg gtt gcc atg gca act cca240 Thr Phe Gly Thr Glu Lys Ala Ile Leu Leu Val Ala Met Ala Thr Pro 6570 75 80 gag gat atg aga atc aat gca gaa cat atc aga ata gcc gat caa ttt288 Glu Asp Met Arg Ile Asn Ala Glu His Ile Arg Ile Ala Asp Gln Phe 8590 95 gtg gaa gta cct ggt ggg acc aat aac aat aac tac gcc aat gtg cag336 Val Glu Val Pro Gly Gly Thr Asn Asn Asn Asn Tyr Ala Asn Val Gln 100105 110 ctt att cta gag att gct gag ata act cac gtt gat gcg gtg tgg cct384 Leu Ile Leu Glu Ile Ala Glu Ile Thr His Val Asp Ala Val Trp Pro 115120 125 ggt tgg ggt cat gca tca gaa aat cct gag ctt cca gat gca tta aaa432 Gly Trp Gly His Ala Ser Glu Asn Pro Glu Leu Pro Asp Ala Leu Lys 130135 140 gca aag gga att gta ttc ctt gga cct cct gct ata tct atg gca gca480 Ala Lys Gly Ile Val Phe Leu Gly Pro Pro Ala Ile Ser Met Ala Ala 145150 155 160 ttg gga gac aaa att ggt tcc tcg ttg att gct cag gca gca gaagtt 528 Leu Gly Asp Lys Ile Gly Ser Ser Leu Ile Ala Gln Ala Ala Glu Val165 170 175 cca acc ctt cca tgg agt ggt tct cat gtg aaa att cct cca gaaagt 576 Pro Thr Leu Pro Trp Ser Gly Ser His Val Lys Ile Pro Pro Glu Ser180 185 190 gac ttg att act att cct gat gaa att tac cgt gca gca tgt gtttat 624 Asp Leu Ile Thr Ile Pro Asp Glu Ile Tyr Arg Ala Ala Cys Val Tyr195 200 205 aca aca gaa gaa gca att gca agt tgt caa gta gta ggt tac cctgca 672 Thr Thr Glu Glu Ala Ile Ala Ser Cys Gln Val Val Gly Tyr Pro Ala210 215 220 atg att aag gca tct tgg ggt ggt ggc ggc aaa ggc ata aga aaggtt 720 Met Ile Lys Ala Ser Trp Gly Gly Gly Gly Lys Gly Ile Arg Lys Val225 230 235 240 cat aat gat gat gag gtt agg gca ttg ttc aag caa gtt caaggt gaa 768 His Asn Asp Asp Glu Val Arg Ala Leu Phe Lys Gln Val Gln GlyGlu 245 250 255 gta cca ggc tca cct ata ttt ata atg aaa gtt gct tcc cagagc cga 816 Val Pro Gly Ser Pro Ile Phe Ile Met Lys Val Ala Ser Gln SerArg 260 265 270 cat ctt gaa gtc caa ttg att tgc gat cag cac gga aat tttgca gca 864 His Leu Glu Val Gln Leu Ile Cys Asp Gln His Gly Asn Phe AlaAla 275 280 285 ttg cac agc cgt gat tgt agt gtt caa aga agg cat caa aagatt att 912 Leu His Ser Arg Asp Cys Ser Val Gln Arg Arg His Gln Lys IleIle 290 295 300 gaa gag ggt ccc att act gta gca cct cca gaa acg gtg aaagaa ctt 960 Glu Glu Gly Pro Ile Thr Val Ala Pro Pro Glu Thr Val Lys GluLeu 305 310 315 320 gaa cag gcg gct aga aga tta gct aaa tct gta aat tatgtg ggg gca 1008 Glu Gln Ala Ala Arg Arg Leu Ala Lys Ser Val Asn Tyr ValGly Ala 325 330 335 gct acc gtt gag tat ctt tat agc atg gaa act ggc gagtac tac ttt 1056 Ala Thr Val Glu Tyr Leu Tyr Ser Met Glu Thr Gly Glu TyrTyr Phe 340 345 350 tta gag ttg aac ccc cga cta cag gtt gag cat cct gttact gaa tgg 1104 Leu Glu Leu Asn Pro Arg Leu Gln Val Glu His Pro Val ThrGlu Trp 355 360 365 ata gct gag ata aat ctg cca gca gca caa gtt gca gttggg atg ggc 1152 Ile Ala Glu Ile Asn Leu Pro Ala Ala Gln Val Ala Val GlyMet Gly 370 375 380 atc cca ctc tgg caa att cct gag att agg cgt ttc tatggg atg gaa 1200 Ile Pro Leu Trp Gln Ile Pro Glu Ile Arg Arg Phe Tyr GlyMet Glu 385 390 395 400 cat ggt ggg gga aat gat ggt tgg aag aaa aca tcagtg tta gct acc 1248 His Gly Gly Gly Asn Asp Gly Trp Lys Lys Thr Ser ValLeu Ala Thr 405 410 415 cct ttt gat ttt gac gaa gca caa tct aca aag ccgaaa ggt cat tgt 1296 Pro Phe Asp Phe Asp Glu Ala Gln Ser Thr Lys Pro LysGly His Cys 420 425 430 gtg gct gta cga gtc acc agt gag gac ccc gat gatggt ttt acg cct 1344 Val Ala Val Arg Val Thr Ser Glu Asp Pro Asp Asp GlyPhe Thr Pro 435 440 445 aca gga gga aaa gtg cag gag ctc agc ttt aaa agcaag cca aat gtg 1392 Thr Gly Gly Lys Val Gln Glu Leu Ser Phe Lys Ser LysPro Asn Val 450 455 460 tgg gct tat ttc tct gtt aag tcc gga gga gga attcat gaa ttc tca 1440 Trp Ala Tyr Phe Ser Val Lys Ser Gly Gly Gly Ile HisGlu Phe Ser 465 470 475 480 gat tct caa ttt gga cat gtt ttt gcg ttt ggagaa tct aga gct tta 1488 Asp Ser Gln Phe Gly His Val Phe Ala Phe Gly GluSer Arg Ala Leu 485 490 495 gca att gca aat atg gta ctg ggg ttg aag gaaatt caa att cga gga 1536 Ala Ile Ala Asn Met Val Leu Gly Leu Lys Glu IleGln Ile Arg Gly 500 505 510 gaa att cgt acc aac gtt gat tac aca att gatctt ctg aat gct tca 1584 Glu Ile Arg Thr Asn Val Asp Tyr Thr Ile Asp LeuLeu Asn Ala Ser 515 520 525 gac tac aga gac aac aaa att cac aca gga tggcta gac agt aga att 1632 Asp Tyr Arg Asp Asn Lys Ile His Thr Gly Trp LeuAsp Ser Arg Ile 530 535 540 gca atg cgg gtt aga gca gag agg cct ccc tggtat ctg tct gtt gtt 1680 Ala Met Arg Val Arg Ala Glu Arg Pro Pro Trp TyrLeu Ser Val Val 545 550 555 560 ggt ggg gca ctc tat aaa gct tct gcc agcagt gca gct tta gtt tcg 1728 Gly Gly Ala Leu Tyr Lys Ala Ser Ala Ser SerAla Ala Leu Val Ser 565 570 575 gac tat gtt ggc tat ctt gaa aag ggg caaatc cct ccc aag cac att 1776 Asp Tyr Val Gly Tyr Leu Glu Lys Gly Gln IlePro Pro Lys His Ile 580 585 590 tct ctt gtc cat tct caa gtt tct ttg agcatt gaa gga agc aaa tac 1824 Ser Leu Val His Ser Gln Val Ser Leu Ser IleGlu Gly Ser Lys Tyr 595 600 605 acg att gac atg gta cga gga gga cct ggaagt tac aaa ttg aaa ttg 1872 Thr Ile Asp Met Val Arg Gly Gly Pro Gly SerTyr Lys Leu Lys Leu 610 615 620 aat caa tcg gag ata gaa gcg gag ata cacact tta cgt gat gga ggt 1920 Asn Gln Ser Glu Ile Glu Ala Glu Ile His ThrLeu Arg Asp Gly Gly 625 630 635 640 ttg cta atg cag ttg gat gga aac agtcat gta ata tat gca gag gaa 1968 Leu Leu Met Gln Leu Asp Gly Asn Ser HisVal Ile Tyr Ala Glu Glu 645 650 655 gaa gca gct gga act cgg ctt tta atagat gga agg act tgc ttg ctt 2016 Glu Ala Ala Gly Thr Arg Leu Leu Ile AspGly Arg Thr Cys Leu Leu 660 665 670 cag aat gat gac gat cca tca aag ttaatt gga gag aca ccg tgc aag 2064 Gln Asn Asp Asp Asp Pro Ser Lys Leu IleGly Glu Thr Pro Cys Lys 675 680 685 ctt ctg aga tat ttg gtt gcg gat gatagt cag att gat gca gac aca 2112 Leu Leu Arg Tyr Leu Val Ala Asp Asp SerGln Ile Asp Ala Asp Thr 690 695 700 cca tat gct gaa gtt gag gtc atg aagatg tgc atg cct ctt ctt tcc 2160 Pro Tyr Ala Glu Val Glu Val Met Lys MetCys Met Pro Leu Leu Ser 705 710 715 720 cct gct tct gga att att cat ttcaga atg gct gaa ggt caa gcc atg 2208 Pro Ala Ser Gly Ile Ile His Phe ArgMet Ala Glu Gly Gln Ala Met 725 730 735 cag gct ggt gaa ctt ata gca aagctt gat cta gat gat ggt tct gca 2256 Gln Ala Gly Glu Leu Ile Ala Lys LeuAsp Leu Asp Asp Gly Ser Ala 740 745 750 gta agg aag gca gaa ccc ttc actggg agc ttc cct atc ctg ggc cct 2304 Val Arg Lys Ala Glu Pro Phe Thr GlySer Phe Pro Ile Leu Gly Pro 755 760 765 cct act gca att tca ggt aaa gttcat cag aaa tgt gca gca agc tta 2352 Pro Thr Ala Ile Ser Gly Lys Val HisGln Lys Cys Ala Ala Ser Leu 770 775 780 aac gct gca cgg atg att ctt gctggc tat gag cac aac att gat gaa 2400 Asn Ala Ala Arg Met Ile Leu Ala GlyTyr Glu His Asn Ile Asp Glu 785 790 795 800 gtt gtg gtc aaa agt ttg ctcaat tgc ctt gac agc cct gaa ctg cct 2448 Val Val Val Lys Ser Leu Leu AsnCys Leu Asp Ser Pro Glu Leu Pro 805 810 815 ttc ctt caa tgg caa gag tgcttt gca gtt ttg gca acc cgt ctt ccc 2496 Phe Leu Gln Trp Gln Glu Cys PheAla Val Leu Ala Thr Arg Leu Pro 820 825 830 aaa gat ctt aga aac gag ttggaa gct aaa tat aag gag ttc gaa att 2544 Lys Asp Leu Arg Asn Glu Leu GluAla Lys Tyr Lys Glu Phe Glu Ile 835 840 845 att tca agc tcc caa act attgat ttc cct gcc aaa tta ttg aag gca 2592 Ile Ser Ser Ser Gln Thr Ile AspPhe Pro Ala Lys Leu Leu Lys Ala 850 855 860 atc ctt gaa gct cat ctt tcctcc tgt cct gaa aac gaa aaa gga gcc 2640 Ile Leu Glu Ala His Leu Ser SerCys Pro Glu Asn Glu Lys Gly Ala 865 870 875 880 tta gaa aga cta gtt gaaccg ctg aca agt ctt gta aag tct tat gag 2688 Leu Glu Arg Leu Val Glu ProLeu Thr Ser Leu Val Lys Ser Tyr Glu 885 890 895 ggt gga aga gag agc catgct cat aaa att gtt caa tct cta ttt gaa 2736 Gly Gly Arg Glu Ser His AlaHis Lys Ile Val Gln Ser Leu Phe Glu 900 905 910 gag tat ctt tca gtt gaagaa cta ttc agt gat aat ata cag gct gat 2784 Glu Tyr Leu Ser Val Glu GluLeu Phe Ser Asp Asn Ile Gln Ala Asp 915 920 925 gta att gaa cga ctc cgtctt caa tac aag aaa gat ttg ttg aag att 2832 Val Ile Glu Arg Leu Arg LeuGln Tyr Lys Lys Asp Leu Leu Lys Ile 930 935 940 gta gat att gtg ctc tctcat cag ggt gtc aag agc aaa aac aag ctg 2880 Val Asp Ile Val Leu Ser HisGln Gly Val Lys Ser Lys Asn Lys Leu 945 950 955 960 ata ctg cga cta atggat aaa ctg gtt tac cct aat cct gct gcc tat 2928 Ile Leu Arg Leu Met AspLys Leu Val Tyr Pro Asn Pro Ala Ala Tyr 965 970 975 agg gat caa tta atccga ttc tcc caa ctc aac cat ata gtt tat tct 2976 Arg Asp Gln Leu Ile ArgPhe Ser Gln Leu Asn His Ile Val Tyr Ser 980 985 990 gag ttg gct ctt aaggca agt caa ctg ttg gag caa act aaa ctc agt 3024 Glu Leu Ala Leu Lys AlaSer Gln Leu Leu Glu Gln Thr Lys Leu Ser 995 1000 1005 gaa ctt cga tccagc att gct aga agt ctt tct gaa cta gaa atg ttt 3072 Glu Leu Arg Ser SerIle Ala Arg Ser Leu Ser Glu Leu Glu Met Phe 1010 1015 1020 acc gag gatggt gaa aat att gat act ccg aag agg aag agt gcc att 3120 Thr Glu Asp GlyGlu Asn Ile Asp Thr Pro Lys Arg Lys Ser Ala Ile 1025 1030 1035 1040 aatgac aga atg gag gac ctt gtg agc gct cct ttg gct gtt gaa gat 3168 Asn AspArg Met Glu Asp Leu Val Ser Ala Pro Leu Ala Val Glu Asp 1045 1050 1055gcc ctt gtt ggt tta ttt gat cac agc gat cac acc ctt caa agg aga 3216 AlaLeu Val Gly Leu Phe Asp His Ser Asp His Thr Leu Gln Arg Arg 1060 10651070 gtt gtt gaa act tat atc cgt agg ctc tat cag cca tat ctt gtc aaa3264 Val Val Glu Thr Tyr Ile Arg Arg Leu Tyr Gln Pro Tyr Leu Val Lys1075 1080 1085 gat agc atc agg atg cag tgg cac aga tct ggc ctt att gctaca tgg 3312 Asp Ser Ile Arg Met Gln Trp His Arg Ser Gly Leu Ile Ala ThrTrp 1090 1095 1100 gaa ttc tta gaa gaa tac gtt gaa cgg aag aat ggg gttgaa gac aaa 3360 Glu Phe Leu Glu Glu Tyr Val Glu Arg Lys Asn Gly Val GluAsp Lys 1105 1110 1115 1120 aca ctg gtg gag aaa cat agt gag aag aaa tgggga gtg atg gtt gta 3408 Thr Leu Val Glu Lys His Ser Glu Lys Lys Trp GlyVal Met Val Val 1125 1130 1135 att aaa tct ctt cag ttt ttg cca gca attatc agt gct gca tta aga 3456 Ile Lys Ser Leu Gln Phe Leu Pro Ala Ile IleSer Ala Ala Leu Arg 1140 1145 1150 gaa gca acc aat aac ttt cac gat cctctt aaa agt ggt tct ggt gac 3504 Glu Ala Thr Asn Asn Phe His Asp Pro LeuLys Ser Gly Ser Gly Asp 1155 1160 1165 tca agt aac cat ggt aat atg atgcat att gga tta gtg ggg atc aac 3552 Ser Ser Asn His Gly Asn Met Met HisIle Gly Leu Val Gly Ile Asn 1170 1175 1180 aac caa atg agt tta ctt caagac agt ggt gat gag gat cag gct caa 3600 Asn Gln Met Ser Leu Leu Gln AspSer Gly Asp Glu Asp Gln Ala Gln 1185 1190 1195 1200 gaa aga att gat aagttg gcc aaa ata ctc aga gag cag gaa ata ggg 3648 Glu Arg Ile Asp Lys LeuAla Lys Ile Leu Arg Glu Gln Glu Ile Gly 1205 1210 1215 tcc ata ata catgct gca ggt gtt gga gat att agc tgt atc ata cag 3696 Ser Ile Ile His AlaAla Gly Val Gly Asp Ile Ser Cys Ile Ile Gln 1220 1225 1230 agg gat gaaggg cgt gct cca atg agg cat tcc ttt cac tgg tca tct 3744 Arg Asp Glu GlyArg Ala Pro Met Arg His Ser Phe His Trp Ser Ser 1235 1240 1245 gaa aagcta tat tat gta gag gaa cca ttg ttg ctc cat ctt gaa cct 3792 Glu Lys LeuTyr Tyr Val Glu Glu Pro Leu Leu Leu His Leu Glu Pro 1250 1255 1260 ccccta tcc att tat ctt gaa ctg gac aag ctt aag tgc tat gaa aat 3840 Pro LeuSer Ile Tyr Leu Glu Leu Asp Lys Leu Lys Cys Tyr Glu Asn 1265 1270 12751280 att cgc tat aca cca tcc cga gat cgt caa tgg cac ctc tac aca gtt3888 Ile Arg Tyr Thr Pro Ser Arg Asp Arg Gln Trp His Leu Tyr Thr Val1285 1290 1295 gtg gat acc aag cca caa cca att caa aga atg ttt ctt cgaaca ctt 3936 Val Asp Thr Lys Pro Gln Pro Ile Gln Arg Met Phe Leu Arg ThrLeu 1300 1305 1310 atc aga cag cca acc aca aat gaa gga tac tct tct tatcaa aga ctg 3984 Ile Arg Gln Pro Thr Thr Asn Glu Gly Tyr Ser Ser Tyr GlnArg Leu 1315 1320 1325 gat gca gaa acg tcc cgt acc caa ttg gct atg tcttat act tca agg 4032 Asp Ala Glu Thr Ser Arg Thr Gln Leu Ala Met Ser TyrThr Ser Arg 1330 1335 1340 agc att ttt agg tcc cta atg ggc gca atg gaggag ttg gaa ctt aac 4080 Ser Ile Phe Arg Ser Leu Met Gly Ala Met Glu GluLeu Glu Leu Asn 1345 1350 1355 1360 tca cac aat acc acc atc aaa tct gaacat gct cat atg tac ctc tat 4128 Ser His Asn Thr Thr Ile Lys Ser Glu HisAla His Met Tyr Leu Tyr 1365 1370 1375 atc ata cgc gag cag caa ata gatgat ctt gtg cct tat tcc aag aaa 4176 Ile Ile Arg Glu Gln Gln Ile Asp AspLeu Val Pro Tyr Ser Lys Lys 1380 1385 1390 att aac ata gaa gct ggc caagaa gaa aca aca gtt gag gca atc ttg 4224 Ile Asn Ile Glu Ala Gly Gln GluGlu Thr Thr Val Glu Ala Ile Leu 1395 1400 1405 gaa gaa ctg gca cag gaaatc cat tcc tct gtt ggt gta aga atg cac 4272 Glu Glu Leu Ala Gln Glu IleHis Ser Ser Val Gly Val Arg Met His 1410 1415 1420 aga tta ggc gtt ttcgtg tgg gaa atc aag ctc tgg att aca gca tgt 4320 Arg Leu Gly Val Phe ValTrp Glu Ile Lys Leu Trp Ile Thr Ala Cys 1425 1430 1435 1440 gga cag gcaaat ggt gct tgg agg gtc att gta aac aat gtg act ggt 4368 Gly Gln Ala AsnGly Ala Trp Arg Val Ile Val Asn Asn Val Thr Gly 1445 1450 1455 cat acatgc act gta cat ata tat cga gag atg gag gat gcc acc act 4416 His Thr CysThr Val His Ile Tyr Arg Glu Met Glu Asp Ala Thr Thr 1460 1465 1470 cataaa gtg gtc tac agt tca gtc act gta aag ggt ccg ttg cat ggt 4464 His LysVal Val Tyr Ser Ser Val Thr Val Lys Gly Pro Leu His Gly 1475 1480 1485gta ccg gtg aat gaa aac tat caa cct ttg gga ggt att gac cga aaa 4512 ValPro Val Asn Glu Asn Tyr Gln Pro Leu Gly Gly Ile Asp Arg Lys 1490 14951500 cgt ctt gca gcg aga aag aac agc acc aca tac tgc tat gat ttc ccc4560 Arg Leu Ala Ala Arg Lys Asn Ser Thr Thr Tyr Cys Tyr Asp Phe Pro1505 1510 1515 1520 ctt gca ttt caa aca tcc ttg gaa cag tcc tgg tca atacag cag aca 4608 Leu Ala Phe Gln Thr Ser Leu Glu Gln Ser Trp Ser Ile GlnGln Thr 1525 1530 1535 gga att caa aga gct aat gat aag gat ctc cta aaagta aca gag ctt 4656 Gly Ile Gln Arg Ala Asn Asp Lys Asp Leu Leu Lys ValThr Glu Leu 1540 1545 1550 aaa ttt tcc gaa aaa gct ggt agt tgg ggt acttct ctt gtt cct gca 4704 Lys Phe Ser Glu Lys Ala Gly Ser Trp Gly Thr SerLeu Val Pro Ala 1555 1560 1565 gag cgt ctt cct gga ctc aat gat gtt ggcatg gta gcc tgg ttg atg 4752 Glu Arg Leu Pro Gly Leu Asn Asp Val Gly MetVal Ala Trp Leu Met 1570 1575 1580 gaa atg tgt acg cct aaa ttc cca tctgga agg aca ata ttg gtt gtt 4800 Glu Met Cys Thr Pro Lys Phe Pro Ser GlyArg Thr Ile Leu Val Val 1585 1590 1595 1600 tca aac gat gtg acc ttc aaggcc ggg tct ttt ggc cca aga gag gat 4848 Ser Asn Asp Val Thr Phe Lys AlaGly Ser Phe Gly Pro Arg Glu Asp 1605 1610 1615 gca ttc ttt aga gca gtaact gat ctt gcc tgt gca aag aaa ata cct 4896 Ala Phe Phe Arg Ala Val ThrAsp Leu Ala Cys Ala Lys Lys Ile Pro 1620 1625 1630 tta att tac ttg gcagca aat tct ggt gcc cgt tta ggt gtt gcc gag 4944 Leu Ile Tyr Leu Ala AlaAsn Ser Gly Ala Arg Leu Gly Val Ala Glu 1635 1640 1645 gaa gtc aaa gcttgt ttc aaa gtt ggt tgg tct gag gaa tct aaa cct 4992 Glu Val Lys Ala CysPhe Lys Val Gly Trp Ser Glu Glu Ser Lys Pro 1650 1655 1660 gaa cat ggtttt cag tat gta tat tta aca cct gag gat tat gct cga 5040 Glu His Gly PheGln Tyr Val Tyr Leu Thr Pro Glu Asp Tyr Ala Arg 1665 1670 1675 1680 atcgga tca tca gtg atg gca cat gaa tta aag ctt gaa agt gga gaa 5088 Ile GlySer Ser Val Met Ala His Glu Leu Lys Leu Glu Ser Gly Glu 1685 1690 1695acc aga tgg gtt ata gat acc att gtt ggc aaa gaa gat gga ctg gga 5136 ThrArg Trp Val Ile Asp Thr Ile Val Gly Lys Glu Asp Gly Leu Gly 1700 17051710 gtt gaa aac ttg agt ggt agt ggg gcc att gcc ggt gcc tat tca agg5184 Val Glu Asn Leu Ser Gly Ser Gly Ala Ile Ala Gly Ala Tyr Ser Arg1715 1720 1725 gca tac aag gaa acc ttt aca ttg aca tat gtt acc ggt aggact gtt 5232 Ala Tyr Lys Glu Thr Phe Thr Leu Thr Tyr Val Thr Gly Arg ThrVal 1730 1735 1740 gga att ggt gct tat ctt gct agg ctt ggg atg agg tgcata cag agg 5280 Gly Ile Gly Ala Tyr Leu Ala Arg Leu Gly Met Arg Cys IleGln Arg 1745 1750 1755 1760 ctt gat caa cct ata att ctt acc ggg ttt tcagca tta aac aaa ctt 5328 Leu Asp Gln Pro Ile Ile Leu Thr Gly Phe Ser AlaLeu Asn Lys Leu 1765 1770 1775 ctt ggt agg gag gtg tac agc tct cac atgcaa ctt ggt gga ccg aaa 5376 Leu Gly Arg Glu Val Tyr Ser Ser His Met GlnLeu Gly Gly Pro Lys 1780 1785 1790 atc atg gca aca aat gga gtc gtt catctc aca gtt tcg gac gac ctt 5424 Ile Met Ala Thr Asn Gly Val Val His LeuThr Val Ser Asp Asp Leu 1795 1800 1805 gaa ggc gtt tct tct att ttg aagtgg ctt agc tac gtt cct tct cat 5472 Glu Gly Val Ser Ser Ile Leu Lys TrpLeu Ser Tyr Val Pro Ser His 1810 1815 1820 gta ggt ggt gca ctt ccc attgta aag ccc ctt gat ccc cca gag agg 5520 Val Gly Gly Ala Leu Pro Ile ValLys Pro Leu Asp Pro Pro Glu Arg 1825 1830 1835 1840 gaa gtg gag tat ttaccg gaa aat tca tgc gat cct cgt gct gcc att 5568 Glu Val Glu Tyr Leu ProGlu Asn Ser Cys Asp Pro Arg Ala Ala Ile 1845 1850 1855 tcc gga act ctggat gtt aat gga aag tgg ctg gga ggc att ttt gac 5616 Ser Gly Thr Leu AspVal Asn Gly Lys Trp Leu Gly Gly Ile Phe Asp 1860 1865 1870 aag gac agcttt gtg gag aca cta gaa gga tgg gct aga aca gtt gtt 5664 Lys Asp Ser PheVal Glu Thr Leu Glu Gly Trp Ala Arg Thr Val Val 1875 1880 1885 aca ggaagg gca aag ctt gga gga atc cct gtg gga att gtt gcg gtg 5712 Thr Gly ArgAla Lys Leu Gly Gly Ile Pro Val Gly Ile Val Ala Val 1890 1895 1900 gaaaca caa aca gtt atg caa ata ata cct gct gat cca ggt caa ctt 5760 Glu ThrGln Thr Val Met Gln Ile Ile Pro Ala Asp Pro Gly Gln Leu 1905 1910 19151920 gat tct cac gag agg gtt gtt cct caa gcc ggg cag gtg tgg ttt cct5808 Asp Ser His Glu Arg Val Val Pro Gln Ala Gly Gln Val Trp Phe Pro1925 1930 1935 gat tct gcg acc aag acg gcc caa gcg ata ttg gat ttc aacaga gaa 5856 Asp Ser Ala Thr Lys Thr Ala Gln Ala Ile Leu Asp Phe Asn ArgGlu 1940 1945 1950 gaa ctc cca ctt ttc att atc gca aac tgg aga ggc ttttca ggt gga 5904 Glu Leu Pro Leu Phe Ile Ile Ala Asn Trp Arg Gly Phe SerGly Gly 1955 1960 1965 caa agg gac ctt ttt gaa gga att ctt cag gct ggttcg act att gtg 5952 Gln Arg Asp Leu Phe Glu Gly Ile Leu Gln Ala Gly SerThr Ile Val 1970 1975 1980 gag aac ctt agg aca tac aaa cag ccc ata tttgta tac att cca atg 6000 Glu Asn Leu Arg Thr Tyr Lys Gln Pro Ile Phe ValTyr Ile Pro Met 1985 1990 1995 2000 atg ggt gaa ctc cga ggc ggg gct tgggtt gtt gtc gac agc cga atc 6048 Met Gly Glu Leu Arg Gly Gly Ala Trp ValVal Val Asp Ser Arg Ile 2005 2010 2015 aac tca gac cac att gaa atg tatgct gag cga acg gcc aaa ggt aac 6096 Asn Ser Asp His Ile Glu Met Tyr AlaGlu Arg Thr Ala Lys Gly Asn 2020 2025 2030 gtc ctt gag ccg gaa gga atgatt gaa atc aaa ttt aga aca aga gaa 6144 Val Leu Glu Pro Glu Gly Met IleGlu Ile Lys Phe Arg Thr Arg Glu 2035 2040 2045 ttg ttg gag tgt atg agaaga ctt gat caa caa ttg att aat ttg aag 6192 Leu Leu Glu Cys Met Arg ArgLeu Asp Gln Gln Leu Ile Asn Leu Lys 2050 2055 2060 gaa aaa ctt tct gaagcc aag agt aac aag gac tat ggt gca tat gat 6240 Glu Lys Leu Ser Glu AlaLys Ser Asn Lys Asp Tyr Gly Ala Tyr Asp 2065 2070 2075 2080 tct ctg cagcag cag att aga ttc cgt gag aaa cag ctt ttg cct ttg 6288 Ser Leu Gln GlnGln Ile Arg Phe Arg Glu Lys Gln Leu Leu Pro Leu 2085 2090 2095 tat actcag ata gct aca aaa ttt gct gaa ctc cat gat act tca tta 6336 Tyr Thr GlnIle Ala Thr Lys Phe Ala Glu Leu His Asp Thr Ser Leu 2100 2105 2110 agaatg aaa gca aag ggt gta atc aga gaa gtt ctt gat tgg cgt aag 6384 Arg MetLys Ala Lys Gly Val Ile Arg Glu Val Leu Asp Trp Arg Lys 2115 2120 2125tcg cgt tct gtc ttc tat cag aga ctg cac agg aga atc ggt gag cac 6432 SerArg Ser Val Phe Tyr Gln Arg Leu His Arg Arg Ile Gly Glu His 2130 21352140 tca ctg atc aac atc gtg aga gat gct gct ggt gac caa ttg tca tat6480 Ser Leu Ile Asn Ile Val Arg Asp Ala Ala Gly Asp Gln Leu Ser Tyr2145 2150 2155 2160 gtt tct gcc atg aac ttg ctc aaa gaa tgg tat ctg aattct gat atc 6528 Val Ser Ala Met Asn Leu Leu Lys Glu Trp Tyr Leu Asn SerAsp Ile 2165 2170 2175 gcc aaa ggt aga gaa gat gct tgg ttg gac gat gaagcc ttc ttc aga 6576 Ala Lys Gly Arg Glu Asp Ala Trp Leu Asp Asp Glu AlaPhe Phe Arg 2180 2185 2190 tgg agg gat gat cca gca aac tac gag gat aaacta aag gaa ttg cgc 6624 Trp Arg Asp Asp Pro Ala Asn Tyr Glu Asp Lys LeuLys Glu Leu Arg 2195 2200 2205 gtc cag aga ctg ttg ctt cag ttg aca aatatt ggc gac tcg gct cta 6672 Val Gln Arg Leu Leu Leu Gln Leu Thr Asn IleGly Asp Ser Ala Leu 2210 2215 2220 gat tta caa gct cta cct caa ggt cttgcc gcc ctt tta agc aag ttg 6720 Asp Leu Gln Ala Leu Pro Gln Gly Leu AlaAla Leu Leu Ser Lys Leu 2225 2230 2235 2240 gaa gca tca agt cgc gat aagttg atc agt gaa ctt cgc aaa gta ctc 6768 Glu Ala Ser Ser Arg Asp Lys LeuIle Ser Glu Leu Arg Lys Val Leu 2245 2250 2255 ggt tagtagacag tgaatgctcctgtgatctgc ccatgcactc atgttgtagt 6821 Gly gttcacgtcg ttgatacatgaccatataga aatgtatcca ttttacgatg ttatcatcaa 6881 agtagcagca tccctcggaaaatggacttt cacttgaggg atcaactgta aatgacttcg 6941 gtcttggata gatatttaatttatgcagtt agaggatcat aaccagcatc accatgtttg 7001 gtctatttat ttgctggttgattgattctt tgcgtgtatc tgaataaaca tgtaataatt 7061 tgtaacattg attattttttatgaaaaaca aagttttggg cactcctttt ataaaaaaaa 7121 aaaaaagaat tcctgcagcccgggggatcc 7151 8 2257 PRT Medicago sativa 8 Met Ala Ser Val Gly Arg GlyAsn Gly Tyr Leu Asn Ser Val Leu Pro 1 5 10 15 Ser Arg His Pro Ala ThrThr Thr Glu Val Asp Glu Tyr Cys Asn Ala 20 25 30 Leu Gly Gly Asn Lys ProIle His Ser Ile Leu Ile Ala Asn Asn Gly 35 40 45 Met Ala Ala Val Lys PheIle Arg Ser Val Arg Ser Trp Ala Tyr Glu 50 55 60 Thr Phe Gly Thr Glu LysAla Ile Leu Leu Val Ala Met Ala Thr Pro 65 70 75 80 Glu Asp Met Arg IleAsn Ala Glu His Ile Arg Ile Ala Asp Gln Phe 85 90 95 Val Glu Val Pro GlyGly Thr Asn Asn Asn Asn Tyr Ala Asn Val Gln 100 105 110 Leu Ile Leu GluIle Ala Glu Ile Thr His Val Asp Ala Val Trp Pro 115 120 125 Gly Trp GlyHis Ala Ser Glu Asn Pro Glu Leu Pro Asp Ala Leu Lys 130 135 140 Ala LysGly Ile Val Phe Leu Gly Pro Pro Ala Ile Ser Met Ala Ala 145 150 155 160Leu Gly Asp Lys Ile Gly Ser Ser Leu Ile Ala Gln Ala Ala Glu Val 165 170175 Pro Thr Leu Pro Trp Ser Gly Ser His Val Lys Ile Pro Pro Glu Ser 180185 190 Asp Leu Ile Thr Ile Pro Asp Glu Ile Tyr Arg Ala Ala Cys Val Tyr195 200 205 Thr Thr Glu Glu Ala Ile Ala Ser Cys Gln Val Val Gly Tyr ProAla 210 215 220 Met Ile Lys Ala Ser Trp Gly Gly Gly Gly Lys Gly Ile ArgLys Val 225 230 235 240 His Asn Asp Asp Glu Val Arg Ala Leu Phe Lys GlnVal Gln Gly Glu 245 250 255 Val Pro Gly Ser Pro Ile Phe Ile Met Lys ValAla Ser Gln Ser Arg 260 265 270 His Leu Glu Val Gln Leu Ile Cys Asp GlnHis Gly Asn Phe Ala Ala 275 280 285 Leu His Ser Arg Asp Cys Ser Val GlnArg Arg His Gln Lys Ile Ile 290 295 300 Glu Glu Gly Pro Ile Thr Val AlaPro Pro Glu Thr Val Lys Glu Leu 305 310 315 320 Glu Gln Ala Ala Arg ArgLeu Ala Lys Ser Val Asn Tyr Val Gly Ala 325 330 335 Ala Thr Val Glu TyrLeu Tyr Ser Met Glu Thr Gly Glu Tyr Tyr Phe 340 345 350 Leu Glu Leu AsnPro Arg Leu Gln Val Glu His Pro Val Thr Glu Trp 355 360 365 Ile Ala GluIle Asn Leu Pro Ala Ala Gln Val Ala Val Gly Met Gly 370 375 380 Ile ProLeu Trp Gln Ile Pro Glu Ile Arg Arg Phe Tyr Gly Met Glu 385 390 395 400His Gly Gly Gly Asn Asp Gly Trp Lys Lys Thr Ser Val Leu Ala Thr 405 410415 Pro Phe Asp Phe Asp Glu Ala Gln Ser Thr Lys Pro Lys Gly His Cys 420425 430 Val Ala Val Arg Val Thr Ser Glu Asp Pro Asp Asp Gly Phe Thr Pro435 440 445 Thr Gly Gly Lys Val Gln Glu Leu Ser Phe Lys Ser Lys Pro AsnVal 450 455 460 Trp Ala Tyr Phe Ser Val Lys Ser Gly Gly Gly Ile His GluPhe Ser 465 470 475 480 Asp Ser Gln Phe Gly His Val Phe Ala Phe Gly GluSer Arg Ala Leu 485 490 495 Ala Ile Ala Asn Met Val Leu Gly Leu Lys GluIle Gln Ile Arg Gly 500 505 510 Glu Ile Arg Thr Asn Val Asp Tyr Thr IleAsp Leu Leu Asn Ala Ser 515 520 525 Asp Tyr Arg Asp Asn Lys Ile His ThrGly Trp Leu Asp Ser Arg Ile 530 535 540 Ala Met Arg Val Arg Ala Glu ArgPro Pro Trp Tyr Leu Ser Val Val 545 550 555 560 Gly Gly Ala Leu Tyr LysAla Ser Ala Ser Ser Ala Ala Leu Val Ser 565 570 575 Asp Tyr Val Gly TyrLeu Glu Lys Gly Gln Ile Pro Pro Lys His Ile 580 585 590 Ser Leu Val HisSer Gln Val Ser Leu Ser Ile Glu Gly Ser Lys Tyr 595 600 605 Thr Ile AspMet Val Arg Gly Gly Pro Gly Ser Tyr Lys Leu Lys Leu 610 615 620 Asn GlnSer Glu Ile Glu Ala Glu Ile His Thr Leu Arg Asp Gly Gly 625 630 635 640Leu Leu Met Gln Leu Asp Gly Asn Ser His Val Ile Tyr Ala Glu Glu 645 650655 Glu Ala Ala Gly Thr Arg Leu Leu Ile Asp Gly Arg Thr Cys Leu Leu 660665 670 Gln Asn Asp Asp Asp Pro Ser Lys Leu Ile Gly Glu Thr Pro Cys Lys675 680 685 Leu Leu Arg Tyr Leu Val Ala Asp Asp Ser Gln Ile Asp Ala AspThr 690 695 700 Pro Tyr Ala Glu Val Glu Val Met Lys Met Cys Met Pro LeuLeu Ser 705 710 715 720 Pro Ala Ser Gly Ile Ile His Phe Arg Met Ala GluGly Gln Ala Met 725 730 735 Gln Ala Gly Glu Leu Ile Ala Lys Leu Asp LeuAsp Asp Gly Ser Ala 740 745 750 Val Arg Lys Ala Glu Pro Phe Thr Gly SerPhe Pro Ile Leu Gly Pro 755 760 765 Pro Thr Ala Ile Ser Gly Lys Val HisGln Lys Cys Ala Ala Ser Leu 770 775 780 Asn Ala Ala Arg Met Ile Leu AlaGly Tyr Glu His Asn Ile Asp Glu 785 790 795 800 Val Val Val Lys Ser LeuLeu Asn Cys Leu Asp Ser Pro Glu Leu Pro 805 810 815 Phe Leu Gln Trp GlnGlu Cys Phe Ala Val Leu Ala Thr Arg Leu Pro 820 825 830 Lys Asp Leu ArgAsn Glu Leu Glu Ala Lys Tyr Lys Glu Phe Glu Ile 835 840 845 Ile Ser SerSer Gln Thr Ile Asp Phe Pro Ala Lys Leu Leu Lys Ala 850 855 860 Ile LeuGlu Ala His Leu Ser Ser Cys Pro Glu Asn Glu Lys Gly Ala 865 870 875 880Leu Glu Arg Leu Val Glu Pro Leu Thr Ser Leu Val Lys Ser Tyr Glu 885 890895 Gly Gly Arg Glu Ser His Ala His Lys Ile Val Gln Ser Leu Phe Glu 900905 910 Glu Tyr Leu Ser Val Glu Glu Leu Phe Ser Asp Asn Ile Gln Ala Asp915 920 925 Val Ile Glu Arg Leu Arg Leu Gln Tyr Lys Lys Asp Leu Leu LysIle 930 935 940 Val Asp Ile Val Leu Ser His Gln Gly Val Lys Ser Lys AsnLys Leu 945 950 955 960 Ile Leu Arg Leu Met Asp Lys Leu Val Tyr Pro AsnPro Ala Ala Tyr 965 970 975 Arg Asp Gln Leu Ile Arg Phe Ser Gln Leu AsnHis Ile Val Tyr Ser 980 985 990 Glu Leu Ala Leu Lys Ala Ser Gln Leu LeuGlu Gln Thr Lys Leu Ser 995 1000 1005 Glu Leu Arg Ser Ser Ile Ala ArgSer Leu Ser Glu Leu Glu Met Phe 1010 1015 1020 Thr Glu Asp Gly Glu AsnIle Asp Thr Pro Lys Arg Lys Ser Ala Ile 1025 1030 1035 1040 Asn Asp ArgMet Glu Asp Leu Val Ser Ala Pro Leu Ala Val Glu Asp 1045 1050 1055 AlaLeu Val Gly Leu Phe Asp His Ser Asp His Thr Leu Gln Arg Arg 1060 10651070 Val Val Glu Thr Tyr Ile Arg Arg Leu Tyr Gln Pro Tyr Leu Val Lys1075 1080 1085 Asp Ser Ile Arg Met Gln Trp His Arg Ser Gly Leu Ile AlaThr Trp 1090 1095 1100 Glu Phe Leu Glu Glu Tyr Val Glu Arg Lys Asn GlyVal Glu Asp Lys 1105 1110 1115 1120 Thr Leu Val Glu Lys His Ser Glu LysLys Trp Gly Val Met Val Val 1125 1130 1135 Ile Lys Ser Leu Gln Phe LeuPro Ala Ile Ile Ser Ala Ala Leu Arg 1140 1145 1150 Glu Ala Thr Asn AsnPhe His Asp Pro Leu Lys Ser Gly Ser Gly Asp 1155 1160 1165 Ser Ser AsnHis Gly Asn Met Met His Ile Gly Leu Val Gly Ile Asn 1170 1175 1180 AsnGln Met Ser Leu Leu Gln Asp Ser Gly Asp Glu Asp Gln Ala Gln 1185 11901195 1200 Glu Arg Ile Asp Lys Leu Ala Lys Ile Leu Arg Glu Gln Glu IleGly 1205 1210 1215 Ser Ile Ile His Ala Ala Gly Val Gly Asp Ile Ser CysIle Ile Gln 1220 1225 1230 Arg Asp Glu Gly Arg Ala Pro Met Arg His SerPhe His Trp Ser Ser 1235 1240 1245 Glu Lys Leu Tyr Tyr Val Glu Glu ProLeu Leu Leu His Leu Glu Pro 1250 1255 1260 Pro Leu Ser Ile Tyr Leu GluLeu Asp Lys Leu Lys Cys Tyr Glu Asn 1265 1270 1275 1280 Ile Arg Tyr ThrPro Ser Arg Asp Arg Gln Trp His Leu Tyr Thr Val 1285 1290 1295 Val AspThr Lys Pro Gln Pro Ile Gln Arg Met Phe Leu Arg Thr Leu 1300 1305 1310Ile Arg Gln Pro Thr Thr Asn Glu Gly Tyr Ser Ser Tyr Gln Arg Leu 13151320 1325 Asp Ala Glu Thr Ser Arg Thr Gln Leu Ala Met Ser Tyr Thr SerArg 1330 1335 1340 Ser Ile Phe Arg Ser Leu Met Gly Ala Met Glu Glu LeuGlu Leu Asn 1345 1350 1355 1360 Ser His Asn Thr Thr Ile Lys Ser Glu HisAla His Met Tyr Leu Tyr 1365 1370 1375 Ile Ile Arg Glu Gln Gln Ile AspAsp Leu Val Pro Tyr Ser Lys Lys 1380 1385 1390 Ile Asn Ile Glu Ala GlyGln Glu Glu Thr Thr Val Glu Ala Ile Leu 1395 1400 1405 Glu Glu Leu AlaGln Glu Ile His Ser Ser Val Gly Val Arg Met His 1410 1415 1420 Arg LeuGly Val Phe Val Trp Glu Ile Lys Leu Trp Ile Thr Ala Cys 1425 1430 14351440 Gly Gln Ala Asn Gly Ala Trp Arg Val Ile Val Asn Asn Val Thr Gly1445 1450 1455 His Thr Cys Thr Val His Ile Tyr Arg Glu Met Glu Asp AlaThr Thr 1460 1465 1470 His Lys Val Val Tyr Ser Ser Val Thr Val Lys GlyPro Leu His Gly 1475 1480 1485 Val Pro Val Asn Glu Asn Tyr Gln Pro LeuGly Gly Ile Asp Arg Lys 1490 1495 1500 Arg Leu Ala Ala Arg Lys Asn SerThr Thr Tyr Cys Tyr Asp Phe Pro 1505 1510 1515 1520 Leu Ala Phe Gln ThrSer Leu Glu Gln Ser Trp Ser Ile Gln Gln Thr 1525 1530 1535 Gly Ile GlnArg Ala Asn Asp Lys Asp Leu Leu Lys Val Thr Glu Leu 1540 1545 1550 LysPhe Ser Glu Lys Ala Gly Ser Trp Gly Thr Ser Leu Val Pro Ala 1555 15601565 Glu Arg Leu Pro Gly Leu Asn Asp Val Gly Met Val Ala Trp Leu Met1570 1575 1580 Glu Met Cys Thr Pro Lys Phe Pro Ser Gly Arg Thr Ile LeuVal Val 1585 1590 1595 1600 Ser Asn Asp Val Thr Phe Lys Ala Gly Ser PheGly Pro Arg Glu Asp 1605 1610 1615 Ala Phe Phe Arg Ala Val Thr Asp LeuAla Cys Ala Lys Lys Ile Pro 1620 1625 1630 Leu Ile Tyr Leu Ala Ala AsnSer Gly Ala Arg Leu Gly Val Ala Glu 1635 1640 1645 Glu Val Lys Ala CysPhe Lys Val Gly Trp Ser Glu Glu Ser Lys Pro 1650 1655 1660 Glu His GlyPhe Gln Tyr Val Tyr Leu Thr Pro Glu Asp Tyr Ala Arg 1665 1670 1675 1680Ile Gly Ser Ser Val Met Ala His Glu Leu Lys Leu Glu Ser Gly Glu 16851690 1695 Thr Arg Trp Val Ile Asp Thr Ile Val Gly Lys Glu Asp Gly LeuGly 1700 1705 1710 Val Glu Asn Leu Ser Gly Ser Gly Ala Ile Ala Gly AlaTyr Ser Arg 1715 1720 1725 Ala Tyr Lys Glu Thr Phe Thr Leu Thr Tyr ValThr Gly Arg Thr Val 1730 1735 1740 Gly Ile Gly Ala Tyr Leu Ala Arg LeuGly Met Arg Cys Ile Gln Arg 1745 1750 1755 1760 Leu Asp Gln Pro Ile IleLeu Thr Gly Phe Ser Ala Leu Asn Lys Leu 1765 1770 1775 Leu Gly Arg GluVal Tyr Ser Ser His Met Gln Leu Gly Gly Pro Lys 1780 1785 1790 Ile MetAla Thr Asn Gly Val Val His Leu Thr Val Ser Asp Asp Leu 1795 1800 1805Glu Gly Val Ser Ser Ile Leu Lys Trp Leu Ser Tyr Val Pro Ser His 18101815 1820 Val Gly Gly Ala Leu Pro Ile Val Lys Pro Leu Asp Pro Pro GluArg 1825 1830 1835 1840 Glu Val Glu Tyr Leu Pro Glu Asn Ser Cys Asp ProArg Ala Ala Ile 1845 1850 1855 Ser Gly Thr Leu Asp Val Asn Gly Lys TrpLeu Gly Gly Ile Phe Asp 1860 1865 1870 Lys Asp Ser Phe Val Glu Thr LeuGlu Gly Trp Ala Arg Thr Val Val 1875 1880 1885 Thr Gly Arg Ala Lys LeuGly Gly Ile Pro Val Gly Ile Val Ala Val 1890 1895 1900 Glu Thr Gln ThrVal Met Gln Ile Ile Pro Ala Asp Pro Gly Gln Leu 1905 1910 1915 1920 AspSer His Glu Arg Val Val Pro Gln Ala Gly Gln Val Trp Phe Pro 1925 19301935 Asp Ser Ala Thr Lys Thr Ala Gln Ala Ile Leu Asp Phe Asn Arg Glu1940 1945 1950 Glu Leu Pro Leu Phe Ile Ile Ala Asn Trp Arg Gly Phe SerGly Gly 1955 1960 1965 Gln Arg Asp Leu Phe Glu Gly Ile Leu Gln Ala GlySer Thr Ile Val 1970 1975 1980 Glu Asn Leu Arg Thr Tyr Lys Gln Pro IlePhe Val Tyr Ile Pro Met 1985 1990 1995 2000 Met Gly Glu Leu Arg Gly GlyAla Trp Val Val Val Asp Ser Arg Ile 2005 2010 2015 Asn Ser Asp His IleGlu Met Tyr Ala Glu Arg Thr Ala Lys Gly Asn 2020 2025 2030 Val Leu GluPro Glu Gly Met Ile Glu Ile Lys Phe Arg Thr Arg Glu 2035 2040 2045 LeuLeu Glu Cys Met Arg Arg Leu Asp Gln Gln Leu Ile Asn Leu Lys 2050 20552060 Glu Lys Leu Ser Glu Ala Lys Ser Asn Lys Asp Tyr Gly Ala Tyr Asp2065 2070 2075 2080 Ser Leu Gln Gln Gln Ile Arg Phe Arg Glu Lys Gln LeuLeu Pro Leu 2085 2090 2095 Tyr Thr Gln Ile Ala Thr Lys Phe Ala Glu LeuHis Asp Thr Ser Leu 2100 2105 2110 Arg Met Lys Ala Lys Gly Val Ile ArgGlu Val Leu Asp Trp Arg Lys 2115 2120 2125 Ser Arg Ser Val Phe Tyr GlnArg Leu His Arg Arg Ile Gly Glu His 2130 2135 2140 Ser Leu Ile Asn IleVal Arg Asp Ala Ala Gly Asp Gln Leu Ser Tyr 2145 2150 2155 2160 Val SerAla Met Asn Leu Leu Lys Glu Trp Tyr Leu Asn Ser Asp Ile 2165 2170 2175Ala Lys Gly Arg Glu Asp Ala Trp Leu Asp Asp Glu Ala Phe Phe Arg 21802185 2190 Trp Arg Asp Asp Pro Ala Asn Tyr Glu Asp Lys Leu Lys Glu LeuArg 2195 2200 2205 Val Gln Arg Leu Leu Leu Gln Leu Thr Asn Ile Gly AspSer Ala Leu 2210 2215 2220 Asp Leu Gln Ala Leu Pro Gln Gly Leu Ala AlaLeu Leu Ser Lys Leu 2225 2230 2235 2240 Glu Ala Ser Ser Arg Asp Lys LeuIle Ser Glu Leu Arg Lys Val Leu 2245 2250 2255 Gly 9 20 DNA ArtificialSequence primer for PCR 9 gtaggcaccc tgctactaca 20 10 23 DNA ArtificialSequence primer for PCR 10 catcaggaat agtaatcaag tca 23 11 46 DNAArtificial Sequence representative construct (3′ end) 11 ccttttataaaaaaaaaaaa aagaattcct gcagcccggg ggatcc 46 12 46 DNA Artificial Sequencerepresentative construct (3′ end) 12 ccttttataa aaaaaaaaaa aagaattcctgcagcccggg ggatcc 46

What is claimed is:
 1. A plant containing a recombinant nucleic acidconstruct comprising a nucleic acid encoding a cytosolic ACCase operablylinked to a promoter, wherein said construct lacks a nucleic acidencoding a transit peptide operably linked to said nucleic acid encodingsaid cytosolic ACCase, wherein said plant produces seeds that exhibit astatistically significant increase in oil content as compared to seedsproduced by a corresponding plant lacking said nucleic acid construct.2. The plant of claim 1, wherein said increase in oil content is fromabout 5% to about 25% greater on a dry weight basis.
 3. The plant ofclaim 1, wherein said nucleic acid encodes a plant cytosolic ACCase. 4.The plant of claim 3, wherein said nucleic acid encodes an alfalfacytosolic ACCase.
 5. The plant of claim 1, wherein said nucleic acidencoding said ACCase lacks introns.
 6. The plant of claim 1, whereinsaid promoter is a cauliflower mosaic virus (CaMV) 35S promoter.
 7. Theplant of claim 6, wherein said nucleic acid encoding said cytosolicACCase lacks introns.
 8. The plant of claim 1, wherein said plant is asoybean plant.
 9. The plant of claim 1, wherein said plant is a Brassicaplant.
 10. The plant of claim 9, wherein said plant is selected from thegroup consisting of Brassica napus, Brassica rapa, Brassica juncea,Brassica carinata, Brassica nigra and Brassica oleracea.
 11. Seedsproduced by the plant of claim
 1. 12. Progeny of the plant of claim 1,wherein said progeny produce seeds that exhibit said statisticallysignificant increase in oil content.
 13. A plant containing arecombinant nucleic acid construct comprising a promoter operably linkedto a cytosolic ACCase coding sequence, wherein said cytosolic ACCasecoding sequence lacks introns, wherein said plant produces seeds thatexhibit a statistically significant increase in oil content as comparedto seeds produced by a corresponding plant lacking said nucleic acidconstruct.
 14. The plant of claim 13, wherein said promoter is a CaMV35S promoter.
 15. The plant of claim 13, wherein said promoter isseed-specific.
 16. The plant of claim 13, wherein said construct furthercomprises a nucleic acid encoding a transit peptide operably linked tosaid cytosolic ACCase coding sequence.
 17. A method of producing aplant, comprising: (a) providing a plant comprising a nucleic acidconstruct comprising a nucleic acid encoding a cytosolic ACCase operablylinked to a promoter; and (b) selecting, for at least one generation,progeny plants that produce seeds exhibiting a statistically significantincrease in oil content as compared to seeds produced by a correspondingplant lacking said nucleic acid construct.
 18. The method of claim 17,wherein said increase in oil content is from about 5% to about 25%greater on a dry weight basis.
 19. The method of claim 17, wherein saidnucleic acid encodes a plant cytosolic ACCase.
 20. The method of claim19, wherein said nucleic acid encodes an alfalfa cytosolic ACCase. 21.The method of claim 17, wherein said nucleic acid encoding saidcytosolic ACCase lacks introns.
 22. The method of claim 17, wherein saidpromoter is a CaMV 35S promoter.
 23. The method of claim 17, whereinsaid selecting is for at least three generations.
 24. The method ofclaim 17, wherein said construct further comprises a nucleic acidsequence encoding a transit peptide operably linked to said nucleic acidencoding said cytosolic ACCase.
 25. The method of claim 24, wherein saidnucleic acid encoding said transit peptide encodes a tobacco smallsubunit Rubisco transit peptide.
 26. The method of claim 24, whereinsaid promoter is a CaMV 35S promoter.
 27. The method of claim 26,wherein said nucleic acid encoding said cytosolic ACCase lacks introns.28. The method of claim 17, wherein said construct lacks nucleic acidsequences encoding a transit peptide operably linked to said nucleicacid encoding said cytosolic ACCase.
 29. The method of claim 28, whereinsaid promoter is a CaMV 35S promoter.
 30. The method of claim 29,wherein said nucleic acid encoding said cytosolic ACCase lacks introns.31. The method of claim 17, wherein said plant is a Brassica plant. 32.The method of claim 31, wherein said plant is selected from the groupconsisting of Brassica napus, Brassica rapa, Brassica juncea, Brassicacarinata, Brassica nigra and Brassica oleracea.
 33. A method ofproducing a plant, comprising the steps of: (a) introducing a constructinto one or more plants, said construct comprising a nucleic acidencoding a cytosolic acetyl ACCase operably linked to a promoter,wherein progeny of one or more of said transgenic plants, following atleast one generation of selection, produce seeds that exhibit astatistically significant increase in oil content as compared to seedsproduced by a corresponding plant lacking said nucleic acid encodingsaid ACCase.
 34. A method of increasing the oil content in seeds,comprising the steps of: (a) creating one or more plants containing anucleic acid construct, said nucleic acid construct comprising a nucleicacid encoding a cytosolic ACCase operably linked to a promoter; and (b)selecting progeny of said one or more plants that exhibit astatistically significant increase in oil content in seeds as comparedto seeds produced by a corresponding plant lacking said nucleic acidencoding said ACCase.
 35. The method of claim 34, wherein said selectionstep comprises selecting progeny that contain said nucleic acidconstruct.
 36. A nucleic acid construct comprising a cytosolic ACCasecoding sequence operably linked to a promoter, wherein said constructlacks a nucleic acid encoding a transit peptide operably linked to saidnucleic acid encoding said cytosolic ACCase.
 37. The nucleic acidconstruct of claim 36, wherein said cytosolic ACCase coding sequencelacks introns.
 38. A nucleic acid construct comprising a cytosolicACCase coding sequence operably linked to a promoter, wherein saidcytosolic ACCase coding sequence lacks introns.
 39. The nucleic acidconstruct of claim 38, wherein said construct further comprises anucleic acid sequence encoding a transit peptide operably linked to saidnucleic acid encoding said cytosolic ACCase.